U.S. patent application number 09/966422 was filed with the patent office on 2003-03-06 for novel human g-protein coupled receptor, hgprbmy6, expressed highly in small intestine.
Invention is credited to Barber, Lauren, Cacace, Angela, Feder, John N., Hawken, Donald R., Kornacker, Michael G., Mintier, Gabe, Ramanathan, Chandra S..
Application Number | 20030044892 09/966422 |
Document ID | / |
Family ID | 27398746 |
Filed Date | 2003-03-06 |
United States Patent
Application |
20030044892 |
Kind Code |
A1 |
Feder, John N. ; et
al. |
March 6, 2003 |
Novel human G-protein coupled receptor, HGPRBMY6, expressed highly
in small intestine
Abstract
The present invention describes a newly discovered human
G-protein coupled receptor and its encoding polynucleotide. Also
described are expression vectors, host cells, agonists,
antagonists, aritisense molecules, and antibodies associated with
the polynucleotide and/or polypeptide of the present invention. In
addition, methods for treating, diagnosing, preventing and
screening for disorders associated with aberrant cell growth and
those related to the small intestine and colon are illustrated.
Inventors: |
Feder, John N.; (Belle Mead,
NJ) ; Mintier, Gabe; (Hightstown, NJ) ;
Ramanathan, Chandra S.; (Wallingford, CT) ; Hawken,
Donald R.; (Lawrenceville, NJ) ; Cacace, Angela;
(Clinton, CT) ; Barber, Lauren; (Griswold, CT)
; Kornacker, Michael G.; (Princeton, NJ) |
Correspondence
Address: |
STEPHEN B. DAVIS
BRISTOL-MYERS SQUIBB COMPANY
PATENT DEPARTMENT
P O BOX 4000
PRINCETON
NJ
08543-4000
US
|
Family ID: |
27398746 |
Appl. No.: |
09/966422 |
Filed: |
September 26, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60315412 |
Aug 28, 2001 |
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60306604 |
Jul 19, 2001 |
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60235602 |
Sep 27, 2000 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 435/6.16; 530/350; 536/23.5 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 15/00 20180101; A61P 1/00 20180101; C07K 14/705 20130101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325; 435/6; 530/350; 536/23.5 |
International
Class: |
C12Q 001/68; C07H
021/04; C07K 014/705; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule consisting of a polynucleotide
having a nucleotide sequence selected from the group consisting of:
a) a polynucleotide fragment of SEQ ID NO:1 or a polynucleotide
fragment of the cDNA sequence included in ATCC Deposit No:PTA-2677,
which is hybridizable to SEQ ID NO:1; b) a polynucleotide encoding
a polypeptide fragment of SEQ ID NO:2 or a polypeptide fragment
encoded by the cDNA sequence included in ATCC Deposit No:PTA-2677,
which is hybridizable to SEQ ID NO:1; c) a polynucleotide encoding
a polypeptide domain of SEQ ID NO:2 or a polypeptide domain encoded
by the cDNA sequence included in ATCC Deposit No:PTA-2677, which is
hybridizable to SEQ ID NO:1; d) a polynucleotide encoding a
polypeptide epitope of SEQ ID NO:2 or a polypeptide epitope encoded
by the cDNA sequence included in ATCC Deposit No:PTA-2677, which is
hybridizable to SEQ ID NO:1; e) a polynucleotide encoding a
polypeptide of SEQ ID NO:2 or the cDNA sequence included in ATCC
Deposit No:PTA-2677, which is hybridizable to SEQ ID NO:1, having
biological activity; f) a polynucleotide which is a variant of SEQ
ID NO:1; g) a polynucleotide which is an allelic variant of SEQ ID
NO:1; h) a polynucleotide which encodes a species homologue of the
SEQ ID NO:2; i) a polynucleotide which represents the complimentary
sequence (antisense) of SEQ ID NO:1; j) a polynucleotide
corresponding to nucleotides 4 to 1680 of SEQ ID NO:1; k) a
polynucleotide corresponding to nucleotides 1 to 1680 of SEQ ID
NO:1; or l) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in (a)-(k),
wherein said polynucleotide does not hybridize under stringent
conditions to a nucleic acid molecule having a nucleotide sequence
of only A residues or of only T residues.
2. The isolated nucleic acid molecule of claim 1, wherein the
polynucleotide fragment comprises a nucleotide sequence encoding a
G-protein coupled receptor protein.
3. The isolated nucleic acid molecule of claim 1, wherein the
polynucleotide fragment comprises a nucleotide sequence encoding
the sequence identified as SEQ ID NO:2 or the polypeptide encoded
by the cDNA sequence included in ATCC Deposit No:PTA-2677, which is
hybridizable to SEQ ID NO:1.
4. The isolated nucleic acid molecule of claim 1, wherein the
polynucleotide fragment comprises the entire nucleotide sequence of
SEQ ID NO:1 or the cDNA sequence included in ATCC Deposit
No:PTA-2677, which is hybridizable to SEQ ID NO:1.
5. The isolated nucleic acid molecule of claim 2, wherein the
nucleotide sequence comprises sequential nucleotide deletions from
either the C-terminus or the N-terminus.
6. The isolated nucleic acid molecule of claim 3, wherein the
nucleotide sequence comprises sequential nucleotide deletions from
either the C-terminus or the N-terminus.
7. A recombinant vector comprising the isolated nucleic acid
molecule of claim 1.
8. A method of making a recombinant host cell comprising the
isolated nucleic acid molecule of claim 1.
9. A recombinant host cell produced by the method of claim 8.
10. The recombinant host cell of claim 9 comprising vector
sequences.
11. An isolated polypeptide comprising an amino acid sequence at
least 95% identical to a sequence selected from the group
consisting of: a) a polypeptide fragment of SEQ ID NO:2 or the
encoded sequence included in ATCC Deposit No:PTA-2677; b) a
polypeptide fragment of SEQ ID NO:2 or the encoded sequence
included in ATCC Deposit No:PTA-2677, having biological activity;
c) a polypeptide domain of SEQ ID NO:2 or the encoded sequence
included in ATCC Deposit No:PTA-2677; d) a polypeptide epitope of
SEQ ID NO:2 or the encoded sequence included in ATCC Deposit
No:PTA-2677; e) a full length protein of SEQ ID NO:2 or the encoded
sequence included in ATCC Deposit No:PTA-2677; f) a variant of SEQ
ID NO:2; g) an allelic variant of SEQ ID NO:2; h) a species
homologue of SEQ ID NO:2; or i) a polypeptide corresponding to
amino acids 2 to 560 of SEQ ID NO:2.
12. The isolated polypeptide of claim 11, wherein the fall length
protein comprises sequential amino acid deletions from either the
C-terminus or the N-terminus.
13. An isolated antibody that binds specifically to the isolated
polypeptide of claim 11.
14. A recombinant host cell that expresses the isolated polypeptide
of claim 11.
15. A method of making an isolated polypeptide comprising: a)
culturing the recombinant host cell of claim 14 under conditions
such that said polypeptide is expressed; and b) recovering said
polypeptide.
16. A polypeptide produced by claim 15.
17. A method for preventing, treating, or ameliorating a medical
condition, comprising administering to a mammalian subject a
therapeutically effective amount of the polypeptide of claim 11 or
the polynucleotide of claim 1.
18. A method of diagnosing a pathological condition or a
susceptibility to a pathological condition in a subject comprising:
a) determining the presence or absence of a mutation in the
polynucleotide of claim 1; and b) diagnosing a pathological
condition or a susceptibility to a pathological condition based on
the presence or absence of said mutation.
19. A method of diagnosing a pathological condition or a
susceptibility to a pathological condition in a subject comprising:
a) determining the presence or amount of expression of the
polypeptide of claim 11 in a biological sample; and b) diagnosing a
pathological condition or a susceptibility to a pathological
condition based on the presence or amount of expression of the
polypeptide.
20. A gene corresponding to the cDNA sequence of SEQ ID NO:2.
21. A method of identifying an activity in a biological assay,
wherein the method comprises: a) expressing the HGPRBMY6 sequence
as set forth in SEQ ID NO:2 in a host cell having; and b) measuring
the resulting activity of the expressed HGPRBMY6.
22. A method for identifying a binding partner to the polypeptide
of claim 11 comprising: a) contacting the polypeptide of claim 11
with a binding partner; and b) determining whether the binding
partner effects an activity of the polypeptide.
23. A method of identifying a compound that modulates the
biological activity of HGPRBMY6, or a GPCR, comprising: a)
combining a candidate modulator compound with a host cell
containing a vector according to claim 7, wherein HGPRBMY6 is
expressed by the cell; and b) measuring an effect of the candidate
modulator compound on the activity of the expressed HGPRBMY6.
24. A compound that modulates the biological activity of human
HGPRBMY6 as identified by the method according to claim 21, 22, or
23.
25. The method of claim 22 wherein said binding partner is a
peptide.
26. A method of treating a disease, disorder, or condition related
to the colon, testis, gastrointestinal, or reproductive system,
comprising administering the G-protein coupled receptor polypeptide
or hornologue according to claim 11 in an amount effective to treat
the small intestine-, colon-, or testis-related disorders.
27. The polynucleotide of claim 2, further comprising a
polynucleotide localized in small intestine, colon, testis, or
colon carcinoma cell lines.
28. The polypeptide of claim 11, further comprising a polypeptide
expressed in small intestine, colon, or testis, or colon carcinoma
cell lines.
29. A cell comprising NFAT/CRE and the polypeptide of claim 11.
30. A cell comprising NFAT G alpha 15 and the polypeptide of claim
11.
31. A method of screening for candidate compounds capable of
modulating activity of a G-protein coupled receptor-encoding
polypeptide, comprising: a) contacting a test compound with the
cell of claim 29 or 30; and b) selecting as candidate modulating
compounds those test compounds that modulate activity of the
G-protein coupled receptor polypeptide.
32. The method according to claim 31, wherein the candidate
compounds are agonists or antagonists of G-protein coupled receptor
activity.
33. The method according to claim 32, wherein the candidate
compounds are peptides.
34. The method according to claim 32, wherein the polypeptide
activity is associated with the small intestine, colon, testis, or
colon cancer.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the fields of
pharmacogenomic, diagnostics and patient therapy. More
specifically, the present invention relates to methods of
diagnosing and/or treating diseases involving the Human G-Protein
Coupled Receptor, HGPRBMY6.
BACKGROUND OF THE INVENTION
[0002] It is well established that many medically significant
biological processes are mediated by proteins participating in
signal transduction pathways that involve G-proteins and/or second
messengers, e.g., cAMP (Lefkowitz, Nature, 351:353-354 (1991)).
Herein these proteins are referred to as proteins participating in
pathways with G-proteins or PPG proteins. Some examples of these
proteins include the GPC receptors, such as those for adrenergic
agents and dopamine (Kobilka, B. K., et al., PNAS, 84:46-50 (1987);
Kobilka, B. K., et al., Science, 238:650-656 (1987); Bunzow, J. R.,
et al., Nature, 336:783-787 (1988)), G-proteins themselves,
effector proteins, e.g., phospholipase C, adenylate cyclase, and
phosphodiesterase, and actuator proteins, e.g., protein kinase A
and protein kinase C (Simon, M. I., et al., Science, 252:802-8
(1991)).
[0003] For example, in one form of signal transduction, the effect
of hormone binding is activation of an enzyme, adenylate cyclase,
inside the cell. Enzyme activation by hormones is dependent on the
presence of the nucleotide GTP, and GTP also influences hormone
binding. A G-protein connects the hormone receptors to adenylate
cyclase. G-protein was shown to exchange GTP for bound GDP when
activated by hormone receptors. The GTP-carrying form then binds to
an activated adenylate cyclase. Hydrolysis of GTP to GDP, catalyzed
by the G-protein itself, returns the G-protein to its basal,
inactive form. Thus, the G-protein serves a dual role, as an
intermediate that relays the signal from receptor to effector, and
as a clock that controls the duration of the signal.
[0004] The membrane protein gene superfamily of G-protein coupled
receptors has been characterized as having seven putative
transmembrane domains. The domains are believed to represent
transmembrane a-helices connected by extracellular or cytoplasmic
loops. G-protein coupled receptors include a wide range of
biologically active receptors, such as hormone, viral, growth
factor and neuroreceptors.
[0005] G-protein coupled receptors have been characterized as
including these seven conserved hydrophobic stretches of about 20
to 30 amino acids, connecting at least eight divergent hydrophilic
loops. The G-protein family of coupled receptors includes dopamine
receptors, which bind to neuroleptic drugs, used for treating
psychotic and neurological disorders. Other examples of members of
this family include calcitonin, adrenergic, endothelin, cAMP,
adenosine, muscarinic, acetylcholine, serotonin, histamine,
thrombin, kinin, follicle stimulating hormone, opsins, endothelial
differentiation gene-i receptor, rhodopsins, odorant,
cytomegalovirus receptors, etc.
[0006] Most G-protein coupled receptors have single conserved
cysteine residues in each of the first two extracellular loops
which form disulfide bonds that are believed to stabilize
functional protein structure. The 7 transmembrane regions are
designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 has been
implicated in signal transduction.
[0007] Phosphorylation and lipidation (palmitylation or
farnesylation) of cysteine residues can influence signal
transduction of some G-protein coupled receptors. Most G-protein
coupled receptors contain potential phosphorylation sites within
the third cytoplasmic loop and/or the carboxyl terminus. For
several G-protein coupled receptors, such as the
.beta.-adrenoreceptor, phosphorylation by protein kinase A and/or
specific receptor kinases mediates receptor desensitization.
[0008] For some receptors, the ligand binding sites of G-protein
coupled receptors are believed to comprise a hydrophilic socket
formed by several G-protein coupled receptors transmembrane
domains, which socket is surrounded by hydrophobic residues of the
G-protein coupled receptors. The hydrophilic side of each G-protein
coupled receptor transmembrane helix is postulated to face inward
and form the polar ligand-binding site. TM3 has been implicated in
several G-protein coupled receptors as having a ligand-binding
site, such as including the TM3 aspartate residue. Additionally,
TM5 serines, a TM6 asparagine and TM6 or TM7 phenylalanines or
tyrosines are also implicated in ligand binding.
[0009] G-protein coupled receptors can be intracellularly coupled
by heterotrimeric G-proteins to various intracellular enzymes, ion
channels and transporters (see, Johnson et al., Endoc. Rev.,
10:317-331(1989)). Different G-protein .beta.-subunits
preferentially stimulate particular effectors to modulate various
biological functions in a cell. Phosphorylation of cytoplasmic
residues of G-protein coupled receptors have been identified as an
important mechanism for the regulation of G-protein coupling of
some G-protein coupled receptors. G-protein coupled receptors are
found in numerous sites within a mammalian host.
[0010] G-protein coupled receptors (GPCRs) are one of the largest
receptor superfamilies known. These receptors are biologically
important and malfunction of these receptors results in diseases
such as Alzheimer's, Parkinson, diabetes, dwarfism, color
blindness, retinal pigmentosa and asthma. GPCRs are also involved
in depression, schizophrenia, sleeplessness, hypertension, anxiety,
stress, renal failure and in several other cardiovascular,
metabolic, neural, oncology and immune disorders (F. Horn and G.
Vriend, J. Mol. Med., 76: 464-468 (1998)). They have also been
shown to play a role in HIV infection (Y. Feng et al., Science,
272: 872-877 (1996)). The structure of GPCRs consists of seven
transmembrane helices that are connected by loops. The N-terminus
is always extracellular and C-terminus is intracellular. GPCRs are
involved in signal transduction. The signal is received at the
extracellular N-terminus side. The signal can be an endogenous
ligand, a chemical moiety or light. This signal is then transduced
through the membrane to the cytosolic side where a heterotrimeric
protein G-protein is activated which in turn elicits aresponse (F.
Horn et al., Recept. and Chann., 5: 305-314 (1998)). Ligands,
agonists and antagonists, for these GPCRs are used for therapeutic
purposes.
[0011] The present invention provides a newly-discovered G-protein
coupled receptor protein, which may be involved in cellular growth
properties in the small intestine, as well as in other
gastrointestinal tissues, such as colon, based on its abundance in
the small intestine and colon. This invention also relates to newly
identified polynucleotides, polypeptides encoded by such
polynucleotides, the use of such polynucleotides and polypeptides,
as well as the production of such polynucleotides and polypeptides.
More particularly, the polypeptides of the present invention are
human 7-transmembrane receptors. In addition, the invention also
relates to inhibiting the action of such polypeptides.
SUMMARY OF THE INVENTION
[0012] The present invention describes a novel human member of the
G-protein coupled receptor (GPCR) family (HGPRBMY6). Based on
sequence homology, the protein HGPRBMY6 is a candidate GPCR. The
HGPRBMY6 sequence has been predicted to contain seven transmembrane
domains which is a characteristic structural feature of GPCRs.
HGPRBMY6 is related to latrophilin, alpha-latrotoxin, and CL3
receptors based on sequence similarity. This orphan GPCR is
expressed highly in small intestine and colonic tissues.
[0013] The present invention provides an isolated HGPRBMY6
polynucleotide as depicted in SEQ ID NO:1.
[0014] The present invention also provides the HGPRBMY6 polypeptide
(MW: 63.2 Kd), encoded by the polynucleotide of SEQ ID NO:1 and
having the amino acid sequence of SEQ ID NO:2, or a functional or
biologically active portion thereof.
[0015] The present invention further provides compositions
comprising the HGPRBMY6 polynucleotide sequence, or a fragment
thereof, or the encoded HGPRBMY6 polypeptide, or a fragment or
portion thereof. Also provided by the present invention are
pharmaceutical compositions comprising at least one HGPRBMY6
polypeptide, or a functional portion thereof, wherein the
compositions further comprise a pharmaceutically acceptable
carrier, excipient, or diluent.
[0016] The present invention provides a novel isolated and
substantially purified polynucleotide that encodes the HGPRBMY6
GPCR homologue. In a particular aspect, the polynucleotide
comprises the nucleotide sequence of SEQ ID NO:1. The present
invention also provides a polynucleotide sequence comprising the
complement of SEQ ID NO:1, or variants thereof. In addition, the
present invention features polynucleotide sequences, which
hybridize under moderately stringent or high stringency conditions
to the polynucleotide sequence of SEQ ID NO:1.
[0017] The present invention further provides a nucleic acid
sequence encoding the HGPRBMY6 polypeptide and an antisense of the
nucleic acid sequence, as well as oligonucleotides, fragments, or
portions of the nucleic acid molecule or antisense molecule. Also
provided are expression vectors and host cells comprising
polynucleotides that encode the HGPRBMY6 polypeptide.
[0018] The present invention provides methods for producing a
polypeptide comprising the amino acid sequence depicted in SEQ ID
NO:2, or a fragment thereof, comprising the steps of a) cultivating
a host cell containing an expression vector containing at least a
functional fragment of the polynucleotide sequence encoding the
HGPRBMY6 protein according to this invention under conditions
suitable for the expression of the polynucleotide; and b)
recovering the polypeptide from the host cell.
[0019] Also provided are antibodies, and binding fragments thereof,
which bind specifically to the HGPRBMY6 polypeptide, or an epitope
thereof, for use as therapeutics and diagnostic agents.
[0020] The present invention also provides methods for screening
for agents which modulate HGPRBMY6 polypeptide, e.g., agonists and
antagonists, as well as modulators, e.g., agonists and antagonists,
particularly those that are obtained from the screening methods
described.
[0021] Also provided by the present invention is a substantially
purified antagonist or inhibitor of the polypeptide of SEQ ID NO:2.
In this regard, and by way of example, a purified antibody that
binds to a polypeptide comprising the amino acid sequence of SEQ ID
NO:2 is provided.
[0022] Substantially purified agonists of the G-protein coupled
receptor polypeptide of SEQ ID NO:2 are further provided.
[0023] The present invention provides HGPRBMY6 nucleic acid
sequences, polypeptide, peptides and antibodies for use in the
diagnosis and/or screening of disorders or diseases associated with
expression of the polynucleotide and its encoded polypeptide as
described herein.
[0024] The present invention provides kits for screening and
diagnosis of disorders associated with aberrant or uncontrolled
cellular development and with the expression of the polynucleotide
and its encoded polypeptide as described herein.
[0025] The present invention further provides methods for the
treatment or prevention of cancers, immune disorders, neurological,
small intestine-related, or colon-related disorders, diseases, or
conditions involving administering, to an individual in need of
treatment or prevention, an effective amount of a purified
antagonist of the HGPRBMY6 polypeptide. Due to its elevated
expression in small intestine and colon, the novel GPCR protein of
the present invention is particularly useful in treating or
preventing gastrointestinal disorders, conditions, or diseases.
[0026] The present invention also provides a method for detecting a
polynucleotide that encodes the HGPRBMY6 polypeptide in a
biological sample comprising the steps of: a) hybridizing the
complement of the polynucleotide sequence encoding SEQ ID NO:2 to a
nucleic acid material of a biological sample, thereby forming a
hybridization complex; and b) detecting the hybridization complex,
wherein the presence of the complex correlates with the presence of
a polynucleotide encoding the HGPRBMY6 polypeptide in the
biological sample. The nucleic acid material may be further
amplified by the polymerase chain reaction prior to
hybridization.
[0027] Further objects, features, and advantages of the present
invention will be better understood upon a reading of the detailed
description of the invention when considered in connection with the
accompanying figures/drawings.
[0028] One aspect of the instant invention comprises methods and
compositions to detect and diagnose alterations in the HGPRBMY6
sequence in tissues and cells as they relate to ligand
response.
[0029] The present invention further provides compositions for
diagnosing small intestine- and colon-related disorders and
response to HGPRBMY6 therapy in humans. In accordance with the
invention, the compositions detect an alteration of the normal or
wild type HGPRBMY6 sequence or its expression product in a patient
sample of cells or tissue.
[0030] The present invention further provides diagnostic probes for
diseases and a patient's response to therapy. The probe sequence
comprises the HGPRBMY6 locus polymorphism. The probes can be
constructed of nucleic acids or amino acids.
[0031] The present invention further provides antibodies that
recognize and bind to the HGPRBMY6 protein. Such antibodies can be
either polyclonal or monoclonal. Antibodies that bind to the
HGPRBMY6 protein can be utilized in a variety of diagnostic and
prognostic formats and therapeutic methods.
[0032] The present invention also provides diagnostic kits for the
determination of the nucleotide sequence of human HGPRBMY6 alleles.
The kits are based on amplification-based assays, nucleic acid
probe assays, protein nucleic acid probe assays, antibody assays or
any combination thereof.
[0033] The instant invention also provides methods for detecting
genetic predisposition, susceptibility and response to therapy
related to the small intestines and colon. In accordance with the
invention, the method comprises isolating a human sample, for
example, blood or tissue from adults, children, embryos or fetuses,
and detecting at least one alteration in the wild-type HGPRBMY6
sequence or its expression product from the sample, wherein the
alterations are indicative of genetic predisposition,
susceptibility or altered response to therapy related to the small
intestine and colon.
[0034] In addition, methods for making determinations as to which
drug to administer, dosages, duration of treatment and the like are
provided.
BRIEF DESCRIPTION OF THE FIGURES
[0035] FIG. 1 shows the full length nucleotide sequence of cDNA
clone HGPRBMY6, a human G-protein coupled receptor (SEQ ID
NO:1).
[0036] FIG. 2 shows the amino acid sequence (SEQ ID NO:2) from the
conceptual translation of the full length HGPRBMY6 cDNA
sequence.
[0037] FIG. 3 shows the 5' untranslated sequence of the orphan
receptor, HGPRBMY6 (SEQ ID NO:3).
[0038] FIG. 4 shows the 3' untranslated sequence of the orphan
receptor, HGPRBMY6 (SEQ ID NO:4).
[0039] FIG. 5 shows the predicted transmembrane region of the
HGPRBMY6 protein where the predicted transmembranes, bold-faced and
underlined, correspond to the peaks with scores above 1500.
[0040] FIGS. 6A-6D show the multiple sequence alignment of the
translated sequence of the orphan G-protein coupled receptor,
HGPRBMY6, where the GCG pileup program was used to generate the
alignment with other G-protein coupled receptor sequences. The
blackened areas represent identical amino acids in more than half
of the listed sequences and the grey highlighted areas represent
similar amino acids. As shown in FIGS. 6A-6D, the sequences are
aligned according to their amino acids, where: HGPRBMY6 (SEQ ID
NO:2) is the translated full length HGPRBMY6 cDNA; O88925 (SEQ ID
NO:8) represents the rat form of CL3AB; O88927 (SEQ ID NO:9) is the
rat form of latrophilin 3; Q9Y3K0 (SEQ ID NO:10) is the human form
of DJ287G14.2, a novel seven transmembrane protein; and Q10922 (SEQ
ID NO:11) is a protein, with weak similarity to GPCRs, from C.
elegans.
[0041] FIG. 7 shows the expression profiling of the novel human
orphan GPCR, HGPRBMY6, as described in Example 3.
[0042] FIG. 8 shows the expression profiling of the novel human
orphan GPCR, HGPRBMY6, as described in Table 1 and Example 4.
[0043] FIG. 9 shows the FACS profile for an untrasfected
CHO-NFAT/CRE cell line.
[0044] FIG. 10 shows the overexpression of HGPRBMY6 constitutively
couples through the NFAT/CRE response element.
[0045] FIG. 11 shows the FACS profile for an untransfected cAMP
Response Element.
[0046] FIG. 12 shows the FACS profile describing that HGPRBMY6
couples throug the cAMP Response Element.
[0047] FIG. 13 shows the FACS profile for an untransfected CHO-NFAT
G alpha 15 cell line.
[0048] FIG. 14 shows the overexpression of HGPRBMY6 constitutively
coupled NFAT Response Element via the promiscuous G protein, G
alpha 15.
[0049] FIG. 15 shows expressed HGPRBMY6 localized to the cell
surface.
[0050] FIG. 16 shows representative transfected CHO-NFAT/CRE cell
lines with intermediate and high beta lactamase expression levels
useful in screens to identify HGPRBMY6 agonists and/or
antagonists.
DETAILED DESCRIPTION OF THE INVENTION
[0051] The present invention provides a novel isolated
polynucleotide and encoded polypeptide, the expression of which is
high in small intestine and colonic tissues. This novel polypeptide
is termed herein HGPRBMY6, an acronym for "Human G-Protein coupled
Receptor BMY6". HGPRBMY6 is also referred to as GPCR29.
[0052] Definitions
[0053] The HGPRBMY6 polypeptide (or protein) refers to the amino
acid sequence of substantially purified HGPRBMY6, which may be
obtained from any species, preferably mammalian, and more
preferably, human, and from a variety of sources, including
natural, synthetic, semi-synthetic, or recombinant. Functional
fragments of the HGPRBMY6 polypeptide are also embraced by the
present invention.
[0054] An "agonist" refers to a molecule which, when bound to the
HGPRBMY6 polypeptide, or a functional fragment thereof, increases
or prolongs the duration of the effect of the HGPRBMY6 polypeptide.
Agonists may include proteins, nucleic acids, carbohydrates, or any
other molecules that bind to and modulate the effect of HGPRBMY6
polypeptide. An antagonist refers to a molecule which, when bound
to the HGPRBMY6 polypeptide, or a functional fragment thereof,
decreases the amount or duration of the biological or immunological
activity of HGPRBMY6 polypeptide. "Antagonists" may include
proteins, nucleic acids, carbohydrates, antibodies, or any other
molecules that decrease or reduce the effect of HGPRBMY6
polypeptide.
[0055] "Nucleic acid sequence", as used herein, refers to an
oligonucleotide, nucleotide, or polynucleotide, and fragments or
portions thereof, and to DNA or RNA of genomic or synthetic origin
which may be single- or double-stranded, and represent the sense or
anti-sense strand. By way of non-limiting example, fragments
include nucleic acid sequences that are greater than 20-60
nucleotides in length, and preferably include fragments that are at
least 70-100 nucleotides, or which are at least 1000 nucleotides or
greater in length.
[0056] Similarly, "amino acid sequence" as used herein refers to an
oligopeptide, peptide, polypeptide, or protein sequence, and
fragments or portions thereof, and to naturally occurring or
synthetic molecules. Amino acid sequence fragments are typically
from about 5 to about 30, preferably from about 5 to about 15 amino
acids in length and retain the biological activity or function of
the HGPRBMY6 polypeptide.
[0057] Where "amino acid sequence" is recited herein to refer to an
amino acid sequence of a naturally occurring protein molecule,
"amino acid sequence" and like terms, such as "polypeptide" or
"protein" are not meant to limit the amino acid sequence to the
complete, native amino acid sequence associated with the recited
protein molecule. In addition, the terms HGPRBMY6 polypeptide and
HGPRBMY6 protein are used interchangeably herein to refer to the
encoded product of the HGPRBMY6 nucleic acid sequence of the
present invention.
[0058] A "variant" of the HGPRBMY6 polypeptide refers to an amino
acid sequence that is altered by one or more amino acids. The
variant may have "conservative" changes, wherein a substituted
amino acid has similar structural or chemical properties, e.g.,
replacement of leucine with isoleucine. More rarely, a variant may
have "non-conservative" changes, e.g., replacement of a glycine
with a tryptophan. Minor variations may also include amino acid
deletions or insertions, or both. Guidance in determining which
amino acid residues may be substituted, inserted, or deleted
without abolishing functional biological or immunological activity
may be found using computer programs well known in the art, for
example, DNASTAR software.
[0059] An "allele" or "allelic sequence" is an alternative form of
the HGPRBMY6 nucleic acid sequence. Alleles may result from at
least one mutation in the nucleic acid sequence and may yield
altered mRNAs or polypeptides whose structure or function may or
may not be altered. Any given gene, whether natural or recombinant,
may have none, one, or many allelic forms. Common mutational
changes, which give rise to alleles, are generally ascribed to
natural deletions, additions, or substitutions of nucleotides. Each
of these types of changes may occur alone, or in combination with
the others, one or more times in a given sequence.
[0060] "Altered" nucleic acid sequences encoding HGPRBMY6
polypeptide include nucleic acid sequences containing deletions,
insertions and/or substitutions of different nucleotides resulting
in a polynucleotide that encodes the same or a functionally
equivalent HGPRBMY6 polypeptide. Altered nucleic acid sequences may
further include polymorphisms of the polynucleotide encoding the
HGPRBMY6 polypeptide; such polymorphisms may or may not be readily
detectable using a particular oligonucleotide probe. The encoded
protein may also contain deletions, insertions, or substitutions of
amino acid residues, which produce a silent change and result in a
functionally equivalent HGPRBMY6 protein. Deliberate amino acid
substitutions may be made on the basis of similarity in polarity,
charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the residues, as long as the biological
activity of HGPRBMY6 protein is retained. For example, negatively
charged amino acids may include aspartic acid and glutamic acid;
positively charged amino acids may include lysine and arginine; and
amino acids with uncharged polar head groups having similar
hydrophilicity values may include leucine, isoleucine, and valine;
glycine and alanine; asparagine and glutamine; serine and
threonine; and phenylalanine and tyrosine.
[0061] "Peptide nucleic acid" (PNA) refers to an antisense molecule
or anti-gene agent which comprises an oligonucleotide ("oligo")
linked via an amide bond, similar to the peptide backbone of amino
acid residues. PNAs typically comprise oligos of at least 5
nucleotides linked via amide bonds. PNAs may or may not terminate
in positively charged amino acid residues to enhance binding
affinities to DNA. These small molecules stop transcript elongation
by binding to their complementary strand of nucleic acid (P. E.
Nielsen et al., 1993, Anticancer Drug Des., 8:53-63). PNA may be
pegylated to extend their lifespan in the cell where they
preferentially bind to complementary single stranded DNA and
RNA.
[0062] "Oligonucleotides" or "oligomers" refer to a nucleic acid
sequence, preferably comprising contiguous nucleotides, of at least
about 6 nucleotides to about 60 nucleotides, preferably at least
about 8 to 10 nucleotides in length, more preferably at least about
12 nucleotides in length e.g., about 15 to 35 nucleotides, or about
15 to 25 nucleotides, or about 20 to 35 nucleotides, which can be
typically used in PCR amplification assays, hybridization assays,
or in microarrays. It will be understood that the term
oligonucleotide is substantially equivalent to the terms primer,
probe, or amplimer, as commonly defined in the art. It will also be
appreciated by those skilled in the pertinent art that a longer
oligonucleotide probe, or mixtures of probes, e.g., degenerate
probes, can be used to detect longer, or more complex, nucleic acid
sequences, for example, genomic DNA. In such cases, the probe may
comprise at least 20-200 nucleotides, preferably, at least 30-100
nucleotides, more preferably, 50-100 nucleotides.
[0063] "Amplification" refers to the production of additional
copies of a nucleic acid sequence and is generally carried out
using polymerase chain reaction (PCR) technologies, which are well
known and practiced in the art (see, D. W. Dieffenbach and G. S.
Dveksler, 1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor
Press, Plainview, N.Y.).
[0064] "Microarray" is an array of distinct polynucleotides or
oligonucleotides synthesized on a substrate, such as paper, nylon,
or other type of membrane; filter; chip; glass slide; or any other
type of suitable solid support.
[0065] The term "antisense" refers to nucleotide sequences, and
compositions containing nucleic acid sequences, which are
complementary to a specific DNA or RNA sequence. The term
"antisense strand" is used in reference to a nucleic acid strand
that is complementary to the "sense" strand. Antisense (i.e.,
complementary) nucleic acid molecules include PNA and may be
produced by any method, including synthesis or transcription. Once
introduced into a cell, the complementary nucleotides combine with
natural sequences produced by the cell to form duplexes, which
block either transcription or translation. The designation
"negative" is sometimes used in reference to the antisense strand,
and "positive" is sometimes used in reference to the sense
strand.
[0066] The term "consensus" refers to the sequence that reflects
the most common choice of base or amino acid at each position among
a series of related DNA, RNA or protein sequences. Areas of
particularly good agreement often represent conserved functional
domains.
[0067] A "deletion" refers to a change in either nucleotide or
amino acid sequence and results in the absence of one or more
nucleotides or amino acid residues. By contrast, an "insertion"
(also termed "addition") refers to a change in a nucleotide or
amino acid sequence that results in the addition of one or more
nucleotides or amino acid residues, as compared with the naturally
occurring molecule. A "substitution" refers to the replacement of
one or more nucleotides or amino acids by different nucleotides or
amino acids.
[0068] A "derivative nucleic acid molecule" refers to the chemical
modification of a nucleic acid encoding, or complementary to, the
encoded HGPRBMY6 polypeptide. Such modifications include, for
example, replacement of hydrogen by an alkyl, acyl, or amino group.
A nucleic acid derivative encodes a polypeptide, which retains the
essential biological and/or functional characteristics of the
natural molecule. A derivative polypeptide is one, which is
modified by glycosylation, pegylation, or any similar process that
retains the biological and/or functional or immunological activity
of the polypeptide from which it is derived.
[0069] The term "biologically active", i.e., functional, refers to
a protein or polypeptide or fragment thereof having structural,
regulatory, or biochemical functions of a naturally occurring
molecule. Likewise, "immunologically active" refers to the
capability of the natural, recombinant, or synthetic HGPRBMY6, or
any oligopeptide thereof, to induce a specific immune response in
appropriate animals or cells, for example, to generate antibodies,
and to bind with specific antibodies.
[0070] The term "hybridization" refers to any process by which a
strand of nucleic acid binds with a complementary strand through
base pairing.
[0071] The term "hybridization complex" refers to a complex formed
between two nucleic acid sequences by virtue of the formation of
hydrogen bonds between complementary G and C bases and between
complementary A and T bases. The hydrogen bonds may be further
stabilized by base stacking interactions. The two complementary
nucleic acid sequences hydrogen bond in an anti-parallel
configuration. A hybridization complex may be formed in solution
(e.g., C.sub.0t or R.sub.0t analysis), or between one nucleic acid
sequence present in solution and another nucleic acid sequence
immobilized on a solid support (e.g., membranes, filters, chips,
pins, or glass slides, or any other appropriate substrate to which
cells or their nucleic acids have been affixed).
[0072] The terms "stringency" or "stringent conditions" refer to
the conditions for hybridization as defined by nucleic acid
composition, salt and temperature. These conditions are well known
in the art and may be altered to identify and/or detect identical
or related polynucleotide sequences in a sample. A variety of
equivalent conditions comprising either low, moderate, or high
stringency depend on factors such as the length and nature of the
sequence (DNA, RNA, base composition), reaction milieu (in solution
or immobilized on a solid substrate), nature of the target nucleic
acid (DNA, RNA, base composition), concentration of salts and the
presence or absence of other reaction components (e.g., formamide,
dextran sulfate and/or polyethylene glycol) and reaction
temperature (within a range of from about 5.degree. C. below the
melting temperature of the probe to about 20.degree. C. to
25.degree. C. below the melting temperature). One or more factors
may be varied to generate conditions, either low or high
stringency, that are different from but equivalent to the
aforementioned conditions.
[0073] As will be understood by those of skill in the art, the
stringency of hybridization may be altered in order to identify or
detect identical or related polynucleotide sequences. As will be
further appreciated by the skilled practitioner, the melting
temperature, T.sub.m, can be approximated by the formulas as known
in the art, depending on a number of parameters, such as the length
of the hybrid or probe in number of nucleotides, or hybridization
buffer ingredients and conditions (see, for example, T. Maniatis et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1982 and J. Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1989; Current Protocols in
Molecular Biology, Eds. F. M. Ausubel et al., Vol. 1, "Preparation
and Analysis of DNA", John Wiley and Sons, Inc., 1994-1995, Suppls.
26, 29, 35 and 42; pp. 2.10.7-2.10.16; G. M. Wahl and S. L. Berger
(1987; Methods Enzymol. 152:399-407); and A. R. Kimmel, 1987;
Methods of Enzymol. 152:507-511). As a general guide, T.sub.m
decreases approximately 1.degree. C.-1.5.degree. C. with every 1%
decrease in sequence homology. Also, in general, the stability of a
hybrid is a function of sodium ion concentration and temperature.
Typically, the hybridization reaction is initially performed under
conditions of low stringency, followed by washes of varying, but
higher stringency. Reference to hybridization stringency, e.g.,
high, moderate, or low stringency, typically relates to such
washing conditions.
[0074] Thus, by way of non-limiting example, "high stringency"
refers to conditions that permit hybridization of those nucleic
acid sequences that form stable hybrids in 0.018M NaCl at about
65.degree. C. (i.e., if a hybrid is not stable in 0.018M NaCl at
about 65.degree. C., it will not be stable under high stringency
conditions). High stringency conditions can be provided, for
instance, by hybridization in 50% formamide, 5.times.Denhardt's
solution, 5.times.SSPE (saline sodium phosphate EDTA) (1.times.SSPE
buffer comprises 0.15 M NaCl, 10 mM Na.sub.2HPO.sub.4, 1 mM EDTA),
(or 1.times.SSC buffer containing 150 mM NaCl, 15 mM Na.sub.3
citrate.2 H.sub.2O, pH 7.0), 0.2% SDS at about 42.degree. C.,
followed by washing in 1.times.SSPE (or saline sodium citrate, SSC)
and 0.1% SDS at a temperature of at least about 42.degree. C.,
preferably about 55.degree. C., more preferably about 65.degree.
C.
[0075] "Moderate stringency" refers, by non-limiting example, to
conditions that permit hybridization in 50% formamide,
5.times.Denhardt's solution, 5.times.SSPE (or SSC), 0.2% SDS at
42.degree. C. (to about 50.degree. C.), followed by washing in
0.2.times.SSPE (or SSC) and 0.2% SDS at a temperature of at least
about 42.degree. C., preferably about 55.degree. C., more
preferably about 65.degree. C.
[0076] Low stringency refers, by non-limiting example, to
conditions that permit hybridization in 10% formamide,
5.times.Denhardt's solution, 6.times.SSPE (or SSC), 0.2% SDS at
42.degree. C., followed by washing in 1.times.SSPE (or SSC) and
0.2% SDS at a temperature of about 45.degree. C., preferably about
50.degree. C.
[0077] For additional stringency conditions, see T. Maniatis et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1982). It is to be understood
that the low, moderate and high stringency hybridization/washing
conditions may be varied using a variety of ingredients, buffers
and temperatures well known to and practiced by the skilled
artisan.
[0078] The terms "complementary" or "complementarity" refer to the
natural binding of polynucleotides under permissive salt and
temperature conditions by base-pairing. For example, the sequence
"A-G-T" binds to the complementary sequence "T-C-A".
Complementarity between two single-stranded molecules may be
"partial", in which only some of the nucleic acids bind, or it may
be complete when total complementarity exists between single
stranded molecules. The degree of complementarity between nucleic
acid strands has significant effects on the efficiency and strength
of hybridization between nucleic acid strands. This is of
particular importance in amplification reactions, which depend upon
binding between nucleic acids strands, as well as in the design and
use of PNA molecules.
[0079] The term "homology" refers to a degree of complementarity.
There may be partial homology or complete homology, wherein
complete homology is equivalent to identity. A partially
complementary sequence that at least partially inhibits an
identical sequence from hybridizing to a target nucleic acid is
referred to using the functional term "substantially homologous".
The inhibition of hybridization of the completely complementary
sequence to the target sequence may be examined using a
hybridization assay (e.g., Southern or Northern blot, solution
hybridization and the like) under conditions of low stringency. A
substantially homologous sequence or probe will compete for and
inhibit the binding (i.e., the hybridization) of a completely
homologous sequence or probe to the target sequence under
conditions of low stringency. Nonetheless, conditions of low
stringency do not permit non-specific binding; low stringency
conditions require that the binding of two sequences to one another
be a specific (i.e., selective) interaction. The absence of
non-specific binding may be tested by the use of a second target
sequence which lacks even a partial degree of complementarity
(e.g., less than about 30% identity). In the absence of
non-specific binding, the probe will not hybridize to the second
non-complementary target sequence.
[0080] Those having skill in the art will know how to determine
percent identity between or among sequences using, for example,
algorithms such as those based on the CLUSTALW computer program (J.
D. Thompson et al., 1994, Nucleic Acids Research, 2(22):4673-4680),
or FASTDB, (Brutlag et al., 1990, Comp. App. Biosci., 6:237-245),
as known in the art. Although the FASTDB algorithm typically does
not consider internal non-matching deletions or additions in
sequences, i.e., gaps, in its calculation, this can be corrected
manually to avoid an overestimation of the % identity. CLUSTALW,
however, does take sequence gaps into account in its identity
calculations.
[0081] A "composition comprising a given polynucleotide sequence"
refers broadly to any composition containing the given
polynucleotide sequence. The composition may comprise a dry
formulation or an aqueous solution. Compositions comprising
polynucleotide sequence (SEQ ID NO:1) encoding HGPRBMY6 polypeptide
(SEQ ID NO:2), or fragments thereof, may be employed as
hybridization probes. The probes may be stored in freeze-dried form
and may be in association with a stabilizing agent such as a
carbohydrate. In hybridizations, the probe may be employed in an
aqueous solution containing salts (e.g., NaCl), detergents or
surfactants (e.g., SDS) and other components (e.g., Denhardt's
solution, dry milk, salmon sperm DNA, and the like).
[0082] The term "substantially purified" refers to nucleic acid
sequences or amino acid sequences that are removed from their
natural environment, isolated or separated, and are at least 60%
free, preferably 75% to 85% free, and most preferably 90% or
greater free from other components with which they are naturally
associated.
[0083] The term "sample", or "biological sample", is meant to be
interpreted in its broadest sense. A biological sample suspected of
containing nucleic acid encoding HGPRBMY6 protein, or fragments
thereof, or HGPRBMY6 protein itself, may comprise a body fluid, an
extract from cells or tissue, chromosomes isolated from a cell
(e.g., a spread of metaphase chromosomes), organelle, or membrane
isolated from a cell, a cell, nucleic acid such as genomic DNA (in
solution or bound to a solid support such as for Southern
analysis), RNA (in solution or bound to a solid support such as for
Northern analysis), cDNA (in solution or bound to a solid support),
a tissue, a tissue print and the like.
[0084] "Transformation" refers to a process by which exogenous DNA
enters and changes a recipient cell. It may occur under natural or
artificial conditions using various methods well known in the art.
Transformation may rely on any known method for the insertion of
foreign nucleic acid sequences into a prokaryotic or eukaryotic
host cell. The method is selected based on the type of host cell
being transformed and may include, but is not limited to, viral
infection, electroporation, heat shock, lipofection, and partial
bombardment. Such "transformed" cells include stably transformed
cells in which the inserted DNA is capable of replication either as
an autonomously replicating plasmid or as part of the host
chromosome. Transformed cells also include those cells, which
transiently express the inserted DNA or RNA for limited periods of
time.
[0085] The term "mimetic" refers to a molecule, the structure of
which is developed from knowledge of the structure of HGPRBMY6
protein, or portions thereof, and as such, is able to effect some
or all of the actions of HGPRBMY6 protein.
[0086] The term "portion" with regard to a protein (as in "a
portion of a given protein") refers to fragments or segments of
that protein. The fragments may range in size from four or five
amino acid residues to the entire amino acid sequence minus one
amino acid. Thus, a protein "comprising at least a portion of the
amino acid sequence of SEQ ID NO: 3" encompasses the full-length
human HGPRBMY6 polypeptide, and fragments thereof.
[0087] The term "antibody" refers to intact molecules as well as
fragments thereof, such as Fab, F(ab').sub.2, Fv, which are capable
of binding an epitopic or antigenic determinant. Antibodies that
bind to HGPRBMY6 polypeptides can be prepared using intact
polypeptides or fragments containing small peptides of interest or
prepared recombinantly for use as the immunizing antigen. The
polypeptide or oligopeptide used to immunize an animal can be
derived from the transition of RNA or synthesized chemically, and
can be conjugated to a carrier protein, if desired. Commonly used
carriers that are chemically coupled to peptides include, but are
not limited to, bovine serum albumin (BSA), keyhole limpet
hemocyanin (KLH), and thyroglobulin. The coupled peptide is then
used to immunize the animal (e.g, a mouse, a rat, or a rabbit).
[0088] The term "humanized" antibody refers to antibody molecules
in which amino acids have been replaced in the non-antigen binding
regions in order to more closely resemble a human antibody, while
still retaining the original binding capability, e.g., as described
in U.S. Pat. No. 5,585,089 to C. L. Queen et al.
[0089] The term "antigenic determinant" refers to that portion of a
molecule that makes contact with a particular antibody (i.e., an
epitope). When a protein or fragment of a protein is used to
immunize a host animal, numerous regions of the protein may induce
the production of antibodies which bind specifically to a given
region or three-dimensional structure on the protein; these regions
or structures are referred to an antigenic determinants. An
antigenic determinant may compete with the intact antigen (i.e.,
the immunogen used to elicit the immune response) for binding to an
antibody.
[0090] The terms "specific binding" or "specifically binding" refer
to the interaction between a protein or peptide and a binding
molecule, such as an agonist, an antagonist, or an antibody. The
interaction is dependent upon the presence of a particular
structure (i.e., an antigenic determinant or epitope) of the
protein that is recognized by the binding molecule. For example, if
an antibody is specific for epitope "A", the presence of a protein
containing epitope A (or free, unlabeled A) in a reaction
containing labeled "A" and the antibody will reduce the amount of
labeled A bound to the antibody.
[0091] The term "correlates with expression of a polynucleotide"
indicates that the detection of the presence of ribonucleic acid
that is similar to SEQ ID NO:1 by Northern analysis is indicative
of the presence of mRNA encoding HGPRBMY6 polypeptide (SEQ ID NO:2)
in a sample and thereby correlates with expression of the
transcript from the polynucleotide encoding the protein.
[0092] An "alteration" in the polynucleotide of SEQ ID NO:1
comprises any alteration in the sequence of the polynucleotides
encoding the HGPRBMY6 polypeptide (SEQ ID NO:2), including
deletions, insertions, and point mutations that may be detected
using hybridization assays. Included within this definition is the
detection of alterations to the genomic DNA sequence which encodes
the HGPRBMY6 polypeptide (e.g., by alterations in the pattern of
restriction fragment length polymorphisms capable of hybridizing to
SEQ ID NO:2), the inability of a selected fragment of the
polypeptide of SEQ ID NO:2 to hybridize to a sample of genomic DNA
(e.g., using allele-specific oligonucleotide probes), and improper
or unexpected hybridization, such as hybridization to a locus other
than the normal chromosomal locus for the polynucleotide sequence
encoding the HGPRBMY6 polypeptide (e.g., using fluorescent in situ
hybridization (FISH) to metaphase chromosome spreads).
DESCRIPTION OF THE PRESENT INVENTION
[0093] The present invention provides a novel human member of the
G-protein coupled receptor (GPCR) family (HGPRBMY6). Based on
sequence homology, the protein HGPRBMY6 is a novel human GPCR. This
protein sequence has been predicted to contain seven transmembrane
domains which is a characteristic structural feature of GPCRs.
HGPRBMY6 is related to latrophilin, alpha-latrotoxin, and CL3
receptors based on sequence similarity. This orphan GPCR is
expressed highly in small intestine and colonic tissues. HGPRBMY6
polypeptides and polynucleotides are useful for diagnosing diseases
related to over- and under-expression of HGPRBMY6 proteins by
identifying mutations in the HGPRBMY6 gene using HGPRBMY6 probes,
or determining HGPRBMY6 protein or mRNA expression levels. HGPRBMY6
polypeptides are also useful for screening compounds, which affect
activity of the protein. The invention encompasses the
polynucleotide encoding the HGPRBMY6 polypeptide and the use of the
HGPRBMY6 polynucleotide or polypeptide, or composition in thereof,
the screening, diagnosis, treatment, or prevention of disorders
associated with aberrant or uncontrolled cellular growth and/or
function, such as intestinal bowel disorders, neoplastic diseases
(e.g., cancers and tumors), with particular regard to those
diseases or disorders related to the small intestine and colon,
e.g. intestinal bowel disorders, in addition to immune,
cardiovascular, and neurological disorders. More specifically,
diseases that can be treated with HGPRBMY6 include intestinal bowel
disorders, pain, anorexia, HIV infections, cancers, bulimia,
asthma, Parkinson's disease, acute heart failure, hypotension,
hypertension, osteoporosis, angina pectoris, myocardial infarction,
psychotic, immune, metabolic, cardiovascular and neurological
disorders.
[0094] Nucleic acids encoding human HGPRBMY6 according to the
present invention were first identified in Incyte CloneID: 2206642
from a library obtained from fetal small intestine tissue through a
computer search for amino acid sequence alignments (see Example
1).
[0095] In one of its embodiments, the present invention encompasses
a polypeptide comprising the amino acid sequence of SEQ ID NO:2 as
shown in FIG. 1. The HGPRBMY6 polypeptide is 560 amino acids in
length and shares amino acid sequence homology with the putative
novel seven transmembrane domain protein, DJ287G14.2 (Acc.
No.:Q9Y3K0). The HGPRBMY6 polypeptide shares 27.5% identity and
39.2% similarity with 363 amino acids of the human putative novel
seven transmembrane domain protein, DJ287G14.2, wherein "similar"
amino acids are those which have the same/similar physical
properties and in many cases, the function is conserved with
similar residues. The HGPRBMY6 polypeptide shares 30.6% identity
and 41.7% similarity with the rattus norvegicus (Norway rat) CL3AB
(Acc. No.:O88925); 30.6% identity and 41.9% similarity with the
rattus norvegicus calcium-independent alpha-latrotoxin receptor 3
precursor (LRP3; Acc. No.:O88927); and 29.3% identity and 39.1%
similarity with the caenorhabditis elegans hypothetical 174.3 KD
protein B0286.2 in chromosome II (Acc. No.:Q10922).
[0096] Variants of the HGPRBMY6 polypeptide are also encompassed by
the present invention. A preferred HGPRBMY6 variant has at least 75
to 80%, more preferably at least 85 to 90%, and even more
preferably at least 90% amino acid sequence identity to the amino
acid sequence claimed herein, and which retains at least one
biological, immunological, or other functional characteristic or
activity of HGPRBMY6 polypeptide. Most preferred is a variant
having at least 95% amino acid sequence identity to that of SEQ ID
NO:2.
[0097] In another embodiment, the present invention encompasses
polynucleotides, which encode HGPRBMY6 polypeptide. Accordingly,
any nucleic acid sequence, which encodes the amino acid sequence of
HGPRBMY6 polypeptide, can be used to produce recombinant molecules
that express HGPRBMY6 protein. In a particular embodiment, the
present invention encompasses the HGPRBMY6 polynucleotide
comprising the nucleic acid sequence of SEQ ID NO:2 and as shown in
FIG. 1. More particularly, the present invention provides the
HGPRBMY6 clone, deposited at the American Type Culture Collection
(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 on
Nov. 15, 2000 and under ATCC Accession No. PTA-2677 according to
the terms of the Budapest Treaty.
[0098] As will be appreciated by the skilled practitioner in the
art, the degeneracy of the genetic code results in the production
of a multitude of nucleotide sequences encoding HGPRBMY6
polypeptide. Some of the sequences bear minimal homology to the
nucleotide sequences of any known and naturally occurring gene.
Accordingly, the present invention contemplates each and every
possible variation of nucleotide sequence that could be made by
selecting combinations based on possible codon choices. These
combinations are made in accordance with the standard triplet
genetic code as applied to the nucleotide sequence of naturally
occurring HGPRBMY6, and all such variations are to be considered as
being specifically disclosed.
[0099] Although nucleotide sequences which encode the HGPRBMY6
polypeptide and its variants are preferably capable of hybridizing
to the nucleotide sequence of the naturally occurring HGPRBMY6
polypeptide under appropriately selected conditions of stringency,
it may be advantageous to produce nucleotide sequences encoding the
HGPRBMY6 polypeptide, or its derivatives, which possess a
substantially different codon usage. Codons may be selected to
increase the rate at which expression of the peptide/polypeptide
occurs in a particular prokaryotic or eukaryotic host in accordance
with the frequency with which particular codons are utilized by the
host. Other reasons for substantially altering the nucleotide
sequence encoding the HGPRBMY6 polypeptide, and its derivatives,
without altering the encoded amino acid sequences include the
production of RNA transcripts having more desirable properties,
such as a greater half-life, than transcripts produced from the
naturally occurring sequence.
[0100] The present invention also encompasses production of DNA
sequences, or portions thereof, which encode the HGPRBMY6
polypeptide, and its derivatives, entirely by synthetic chemistry.
After production, the synthetic sequence may be inserted into any
of the many available expression vectors and cell systems using
reagents that are well known and practiced by those in the art.
Moreover, synthetic chemistry may be used to introduce mutations
into a sequence encoding HGPRBMY6 polypeptide, or any fragment
thereof.
[0101] Also encompassed by the present invention are polynucleotide
sequences that are capable of hybridizing to the claimed nucleotide
sequence of HGPRBMY6, such as that shown in SEQ ID NO:1, under
various conditions of stringency. Hybridization conditions are
typically based on the melting temperature (T.sub.m) of the nucleic
acid binding complex or probe (see, G. M. Wahl and S. L. Berger,
1987; Methods Enzymol., 152:399-407 and A. R. Kimmel, 1987; Methods
of Enzymol., 152:507-511), and may be used at a defined stringency.
For example, included in the present invention are sequences
capable of hybridizing under moderately stringent conditions to the
HGPRBMY6 polypeptide sequence of SEQ ID NO:2 and other sequences
which are degenerate to those which encode HGPRBMY6 polypeptide
(e.g., as a non-limiting example: prewashing solution of
2.times.SSC, 0.5% SDS, 1.0 mM EDTA, pH 8.0, and hybridization
conditions of 50.degree. C., 5.times.SSC, overnight.
[0102] The nucleic acid sequence encoding the HGPRBMY6 protein may
be extended utilizing a partial nucleotide sequence and employing
various methods known in the art to detect upstream sequences such
as promoters and regulatory elements. For example, one method,
which may be employed, is restriction-site PCR, which utilizes
universal primers to retrieve unknown sequence adjacent to a known
locus (G. Sarkar, 1993, PCR Methods Applic., 2:318-322). In
particular, genomic DNA is first amplified in the presence of
primer to a linker sequence and a primer specific to the known
region. The amplified sequences are then subjected to a second
round of PCR with the same linker primer and another specific
primer internal to the first one. Products of each round of PCR are
transcribed with an appropriate RNA polymerase and sequenced using
reverse transcriptase.
[0103] Inverse PCR may also be used to amplify or extend sequences
using divergent primers based on a known region or sequence (T.
Triglia et al., 1988, Nucleic Acids Res., 16:8186). The primers may
be designed using OLIGO 4.06 Primer Analysis software (National
Biosciences Inc., Plymouth, Minn.), or another appropriate program,
to be 22-30 nucleotides in length, to have a GC content of 50% or
more, and to anneal to the target sequence at temperatures about
68.degree.-72.degree. C. The method uses several restriction
enzymes to generate a suitable fragment in the known region of a
gene. The fragment is then circularized by intramolecular ligation
and used as a PCR template.
[0104] Another method which may be used is capture PCR which
involves PCR amplification of DNA fragments adjacent to a known
sequence in human and yeast artificial chromosome (YAC) DNA (M.
Lagerstrom et al., 1991, PCR Methods Applic., 1:111-119). In this
method, multiple restriction enzyme digestions and ligations may
also be used to place an engineered double-stranded sequence into
an unknown portion of the DNA molecule before perfonning PCR. J. D.
Parker et al. (1991; Nucleic Acids Res., 19:3055-3060) provide
another method which may be used to retrieve unknown sequences. In
addition, PCR, nested primers, and PROMOTERFINDER libraries can be
used to walk genomic DNA (Clontech, Palo Alto, Calif.). This
process avoids the need to screen libraries and is useful in
finding intron/exon junctions.
[0105] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
Also, random-primed libraries are preferable, since they will
contain more sequences, which contain the 5' regions of genes. The
use of a randomly primed library may be especially preferable for
situations in which an oligo d(T) library does not yield a
full-length cDNA. Genomic libraries may be useful for extension of
sequence into the 5' and 3' non-transcribed regulatory regions.
[0106] The embodiments of the present invention can be practiced
using methods for DNA sequencing which are well known and generally
available in the art. The methods may employ such enzymes as the
Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical
Corp. Cleveland, Ohio), Taq polymerase (PE Biosystems),
thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway,
N.J.), or combinations of recombinant polymerases and proofreading
exonucleases such as the ELONGASE Amplification System marketed by
Life Technologies (Gaithersburg, Md.). Preferably, the process is
automated with machines such as the Hamilton Micro Lab 2200
(Hamilton, Reno, Nev.), Peltier Thermal Cycler (PTC200; M J
Research, Watertown, Mass.) and the ABI Catalyst and 373 and 377
DNA sequencers (PE Biosystems).
[0107] Commercially available capillary electrophoresis systems may
be used to analyze the size or confirm the nucleotide sequence of
sequencing or PCR products. In particular, capillary sequencing may
employ flowable polymers for electrophoretic separation, four
different fluorescent dyes (one for each nucleotide) which are
laser activated, and detection of the emitted wavelengths by a
charge coupled device camera. Output/light intensity may be
converted to electrical signal using appropriate software (e.g.,
GENOTYPER and SEQUENCE NAVIGATOR, PE Biosystems) and the entire
process--from loading of samples to computer analysis and
electronic data display--may be computer controlled. Capillary
electrophoresis is especially preferable for the sequencing of
small pieces of DNA, which might be present in limited amounts in a
particular sample.
[0108] In another embodiment of the present invention,
polynucleotide sequences or fragments thereof which encode HGPRBMY6
polypeptide, or peptides thereof, may be used in recombinant DNA
molecules to direct the expression of HGPRBMY6 polypeptide product,
or fragments or functional equivalents thereof, in appropriate host
cells. Because of the inherent degeneracy of the genetic code,
other DNA sequences, which encode substantially the same or a
functionally equivalent amino acid sequence, may be produced and
these sequences may be used to clone and express HGPRBMY6
protein.
[0109] As will be appreciated by those having skill in the art, it
may be advantageous to produce HGPRBMY6 polypeptide-encoding
nucleotide sequences possessing non-naturally occurring codons. For
example, codons preferred by a particular prokaryotic or eukaryotic
host can be selected to increase the rate of protein expression or
to produce a recombinant RNA transcript having desirable
properties, such as a half-life which is longer than that of a
transcript generated from the naturally occurring sequence.
[0110] The nucleotide sequence of the present invention can be
engineered using methods generally known in the art in order to
alter HGPRBMY6 polypeptide-encoding sequences for a variety of
reasons, including, but not limited to, alterations which modify
the cloning, processing, and/or expression of the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene
fragments and synthetic oligonucleotides may be used to engineer
the nucleotide sequences. For example, site-directed mutagenesis
may be used to insert new restriction sites, alter glycosylation
patterns, change codon preference, produce splice variants, or
introduce mutations, and the like.
[0111] In another embodiment of the present invention, natural,
modified, or recombinant nucleic acid sequences encoding HGPRBMY6
polypeptide may be ligated to a heterologous sequence to encode a
fusion protein. For example, for screening peptide libraries for
inhibitors of HGPRBMY6 activity, it may be useful to encode a
chimeric HGPRBMY6 protein that can be recognized by a commercially
available antibody. A fusion protein may also be engineered to
contain a cleavage site located between the HGPRBMY6
protein-encoding sequence and the heterologous protein sequence, so
that HGPRBMY6 protein may be cleaved and purified away from the
heterologous moiety.
[0112] In another embodiment, sequences encoding HGPRBMY6
polypeptide may be synthesized in whole, or in part, using chemical
methods well known in the art (see, for example, M. H. Caruthers et
al., 1980, Nucl. Acids Res. Symp. Ser., 215-223 and T. Horn et al.,
1980, Nucl. Acids Res. Symp. Ser., 225-232). Alternatively, the
protein itself may be produced using chemical methods to synthesize
the amino acid sequence of HGPRBMY6 polypeptide, or a fragment or
portion thereof. For example, peptide synthesis can be performed
using various solid-phase techniques (J. Y. Roberge et al., 1995,
Science, 269:202-204) and automated synthesis may be achieved, for
example, using the ABI 431A Peptide Synthesizer (PE
Biosystems).
[0113] The newly synthesized peptide can be substantially purified
by preparative high performance liquid chromatography (e.g., T.
Creighton, 1983, Proteins, Structures and Molecular Principles, W.
H. Freeman and Co., New York, N.Y.), by reversed-phase high
performance liquid chromatography, or other purification methods as
are known in the art. The composition of the synthetic peptides may
be confirmed by amino acid analysis or sequencing (e.g., the Edman
degradation procedure; Creighton, supra). In addition, the amino
acid sequence of HGPRBMY6 polypeptide or any portion thereof, may
be altered during direct synthesis and/or combined using chemical
methods with sequences from other proteins, or any part thereof, to
produce a variant polypeptide.
[0114] To express a biologically active HGPRBMY6 polypeptide or
peptide, the nucleotide sequences encoding HGPRBMY6 polypeptide, or
functional equivalents, may be inserted into an appropriate
expression vector, i.e., a vector, which contains the necessary
elements for the transcription and translation of the inserted
coding sequence.
[0115] Methods, which are well known to those skilled in the art,
may be used to construct expression vectors containing sequences
encoding HGPRBMY6 polypeptide and appropriate transcriptional and
translational control elements. These methods include in vitro
recombinant DNA techniques, synthetic techniques, and in vivo
genetic recombination. Such techniques are described in J. Sambrook
et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring
Harbor Press, Plainview, N.Y. and in F. M. Ausubel et al., 1989,
Current Protocols in Molecular Biology, John Wiley & Sons, New
York, N.Y.
[0116] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding HGPRBMY6 polypeptide.
Such expression vector/host systems include, but are not limited
to, microorganisms such as bacteria transformed with recombinant
bacteriophage, plasmid, or cosmid DNA expression vectors; yeast
transformed with yeast expression vectors; insect cell systems
infected with virus expression vectors (e.g., bacculovirus); plant
cell systems transformed with virus expression vectors (e.g.,
cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)), or
with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or
animal cell systems. The host cell employed is not limiting to the
present invention.
[0117] "Control elements" or "regulatory sequences" are those
non-translated regions of the vector, e.g., enhancers, promoters,
5' and 3' untranslated regions, which interact with host cellular
proteins to carry out transcription and translation. Such elements
may vary in their strength and specificity. Depending on the vector
system and host utilized, any number of suitable transcription and
translation elements, including constitutive and inducible
promoters, may be used. For example, when cloning in bacterial
systems, inducible promoters such as the hybrid lacZ promoter of
the BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1
plasmid (Life Technologies), and the like, may be used. The
baculovirus polyhedrin promoter may be used in insect cells.
Promoters or enhancers derived from the genomes of plant cells
(e.g., heat shock, RUBISCO; and storage protein genes), or from
plant viruses (e.g., viral promoters or leader sequences), may be
cloned into the vector. In mammalian cell systems, promoters from
mammalian genes or from mammalian viruses are preferred. If it is
necessary to generate a cell line that contains multiple copies of
the sequence encoding HGPRBMY6, vectors based on SV40 or EBV may be
used with an appropriate selectable marker.
[0118] In bacterial systems, a number of expression vectors may be
selected, depending upon the use intended for the expressed
HGPRBMY6 product. For example, when large quantities of expressed
protein are needed for the induction of antibodies, vectors, which
direct high level expression of fusion proteins that are readily
purified, may be used. Such vectors include, but are not limited
to, the multifunctional E. coli cloning and expression vectors such
as BLUESCRIPT (Stratagene), in which the sequence encoding HGPRBMY6
polypeptide may be ligated into the vector in-frame with sequences
for the amino-terminal Met and the subsequent 7 residues of
.beta.-galactosidase, so that a hybrid protein is produced; pIN
vectors (see, G. Van Heeke and S. M. Schuster, 1989, J. Biol.
Chem., 264:5503-5509); and the like. pGEX vectors (Promega,
Madison, Wis.) may also be used to express foreign polypeptides, as
fusion proteins with glutathione S-transferase (GST). In general,
such fusion proteins are soluble and can be easily purified from
lysed cells by adsorption to glutathione-agarose beads followed by
elution in the presence of free glutathione. Proteins made in such
systems may be designed to include heparin, thrombin, or factor XA
protease cleavage sites so that the cloned polypeptide of interest
can be released from the GST moiety at will.
[0119] In the yeast, Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters such as alpha
factor, alcohol oxidase, and PGH may be used. (For reviews, see F.
M. Ausubel et al., supra, and Grant et al., 1987, Methods Enzymol.,
153:516-544).
[0120] Should plant expression vectors be desired and used, the
expression of sequences encoding HGPRBMY6 polypeptide may be driven
by any of a number of promoters. For example, viral promoters such
as the 35S and 19S promoters of CaMV may be used alone or in
combination with the omega leader sequence from TMV (N. Takamatsu,
1987, EMBO J., 6:307-311). Alternatively, plant promoters such as
the small subunit of RUBISCO, or heat shock promoters, may be used
(G. Coruzzi et al., 1984, EMBO J., 3:1671-1680; R. Broglie et al.,
1984, Science, 224:838-843; and J. Winter et al., 1991, Results
Probl. Cell Differ. 17:85-105). These constructs can be introduced
into plant cells by direct DNA transformation or pathogen-mediated
transfection. Such techniques are described in a number of
generally available reviews (see, for example, S. Hobbs or L. E.
Murry, In: McGraw Hill Yearbook of Science and Technology (1992)
McGraw Hill, New York, N.Y.; pp. 191-196).
[0121] An insect system may also be used to express HGPRBMY6
polypeptide. For example, in one such system, Autographa
californica nuclear polyhedrosis virus (AcNPV) is used as a vector
to express foreign genes in Spodoptera frugiperda cells or in
Trichoplusia larvae. The sequences encoding HGPRBMY6 polypeptide
may be cloned into a non-essential region of the virus such as the
polyhedrin gene and placed under control of the polyhedrin
promoter. Successful insertion of HGPRBMY6 polypeptide will render
the polyhedrin gene inactive and produce recombinant virus lacking
coat protein. The recombinant viruses may then be used to infect,
for example, S. frugiperda cells or Trichoplusia larvae in which
the HGPRBMY6 polypeptide product may be expressed (E. K. Engelhard
et al., 1994, Proc. Nat. Acad. Sci., 91:3224-3227).
[0122] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding HGPRBMY6 polypeptide may be
ligated into an adenovirus transcription/translation complex
containing the late promoter and tripartite leader sequence.
Insertion in a non-essential E1 or E3 region of the viral genome
may be used to obtain a viable virus which is capable of expressing
HGPRBMY6 polypeptide in infected host cells (J. Logan and T. Shenk,
1984, Proc. Natl. Acad. Sci., 81:3655-3659). In addition,
transcription enhancers, such as the Rous sarcoma virus (RSV)
enhancer, may be used to increase expression in mammalian host
cells.
[0123] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding HGPRBMY6 polypeptide.
Such signals include the ATG initiation codon and adjacent
sequences. In cases where sequences encoding HGPRBMY6 polypeptide,
its initiation codon, and upstream sequences are inserted into the
appropriate expression vector, no additional transcriptional or
translational control signals may be needed. However, in cases
where only coding sequence, or a fragment thereof, is inserted,
exogenous translational control signals, including the ATG
initiation codon, should be provided. Furthermore, the initiation
codon should be in the correct reading frame to ensure translation
of the entire insert. Exogenous translational elements and
initiation codons may be of various origins, both natural and
synthetic. The efficiency of expression may be enhanced by the
inclusion of enhancers which are appropriate for the particular
cell system that is used, such as those described in the literature
(D. Scharf et al., 1994, Results Probl. Cell Differ.,
20:125-162).
[0124] Moreover, a host cell strain may be chosen for its ability
to modulate the expression of the inserted sequences or to process
the expressed protein in the desired fashion. Such modifications of
the polypeptide include, but are not limited to, acetylation,
carboxylation, glycosylation, phosphorylation, lipidation, and
acylation. Post-translational processing which cleaves a "prepro"
form of the protein may also be used to facilitate correct
insertion, folding and/or function. Different host cells having
specific cellular machinery and characteristic mechanisms for such
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and
W138) are available from the American Type Culture Collection
(ATCC), American Type Culture Collection (ATCC), 10801 University
Boulevard, Manassas, Va. 20110-2209, and may be chosen to ensure
the correct modification and processing of the foreign protein.
[0125] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines,
which stably express HGPRBMY6 protein, may be transformed using
expression vectors, which may contain viral origins of replication
and/or endogenous expression elements and a selectable marker gene
on the same, or on a separate, vector. Following the introduction
of the vector, cells may be allowed to grow for 1-2 days in an
enriched cell culture medium before they are switched to selective
medium. The purpose of the selectable marker is to confer
resistance to selection, and its presence allows the growth and
recovery of cells, which successfully express the introduced
sequences. Resistant clones of stably transformed cells may be
proliferated using tissue culture techniques appropriate to the
cell type.
[0126] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
Herpes Simplex Virus thymidine kinase (HSV TK), (M. Wigler et al.,
1977, Cell, 11:223-32) and adenine phosphoribosyltransferase (I.
Lowy et al., 1980, Cell, 22:817-23) genes which can be employed in
tk.sup.- or aprt.sup.- cells, respectively. Also, anti-metabolite,
antibiotic or herbicide resistance can be used as the basis for
selection; for example, dhfr, which confers resistance to
methotrexate (M. Wigler et al., 1980, Proc. Natl. Acad. Sci.,
77:3567-70); npt, which confers resistance to the aminoglycosides
neomycin and G-418 (F. Colbere-Garapin et al., 1981, J. Mol. Biol,
150:1-14); and als or pat, which confer resistance to chlorsulfuron
and phosphinotricin acetyltransferase, respectively (Murry, supra).
Additional selectable genes have been described, for example, trpB,
which allows cells to utilize indole in place of tryptophan, or
hisD, which allows cells to utilize histinol in place of histidine
(S. C. Hartman and R. C. Mulligan, 1988, Proc. Natl. Acad. Sci.,
85:8047-51). Recently, the use of visible markers has gained
popularity with such markers as the anthocyanins,
.beta.-glucuronidase and its substrate GUS, and luciferase and its
substrate luciferin, which are widely used not only to identify
transformants, but also to quantify the amount of transient or
stable protein expression that is attributable to a specific vector
system (C. A. Rhodes et al., 1995, Methods Mol. Biol,
55:121-131).
[0127] Although the presence or absence of marker gene expression
suggests that the gene of interest is also present, the presence
and expression of the desired gene of interest may need to be
confirmed. For example, if the nucleic acid sequence encoding
HGPRBMY6 polypeptide is inserted within a marker gene sequence,
recombinant cells containing sequences encoding HGPRBMY6
polypeptide can be identified by the absence of marker gene
function. Alternatively, a marker gene can be placed in tandem with
a sequence encoding HGPRBMY6 polypeptide under the control of a
single promoter. Expression of the marker gene in response to
induction or selection usually indicates co-expression of the
tandem gene.
[0128] Alternatively, host cells, which contain the nucleic acid,
sequence encoding HGPRBMY6 polypeptide and which express HGPRBMY6
polypeptide product may be identified by a variety of procedures
known to those having skill in the art. These procedures include,
but are not limited to, DNA-DNA or DNA-RNA hybridizations and
protein bioassay or immunoassay techniques, including membrane,
solution, or chip based technologies, for the detection and/or
quantification of nucleic acid or protein.
[0129] The presence of polynucleotide sequences encoding HGPRBMY6
polypeptide can be detected by DNA-DNA or DNA-RNA hybridization, or
by amplification using probes or portions or fragments of
polynucleotides encoding HGPRBMY6 polypeptide. Nucleic acid
amplification based assays involve the use of oligonucleotides or
oligomers, based on the sequences encoding HGPRBMY6 polypeptide, to
detect transformants containing DNA or RNA encoding HGPRBMY6
polypeptide.
[0130] A wide variety of labels and conjugation techniques are
known and employed by those skilled in the art and may be used in
various nucleic acid and amino acid assays. Means for producing
labeled hybridization or PCR probes for detecting sequences related
to polynucleotides encoding HGPRBMY6 polypeptide include
oligo-labeling, nick translation, end-labeling, or PCR
amplification using a labeled nucleotide. Alternatively, the
sequences encoding HGPRBMY6 polypeptide, or any portions or
fragments thereof, may be cloned into a vector for the production
of an mRNA probe. Such vectors are known in the art, are
commercially available, and may be used to synthesize RNA probes in
vitro by addition of an appropriate RNA polymerase, such as T7, T3,
or SP(6) and labeled nucleotides. These procedures may be conducted
using a variety of commercially available kits (e.g., Amersham
Pharmacia Biotech, Promega and U.S. Biochemical Corp.). Suitable
reporter molecules or labels which may be used include
radionuclides, enzymes, fluorescent, chemiluminescent, or
chromogenic agents, as well as substrates, cofactors, inhibitors,
magnetic particles, and the like.
[0131] Host cells transformed with nucleotide sequences encoding
HGPRBMY6 protein, or fragments thereof, may be cultured under
conditions suitable for the expression and recovery of the protein
from cell culture. The protein produced by a recombinant cell may
be secreted or contained intracellularly depending on the sequence
and/or the vector used. As will be understood by those having skill
in the art, expression vectors containing polynucleotides which
encode HGPRBMY6 protein may be designed to contain signal sequences
which direct secretion of the HGPRBMY6 protein through a
prokaryotic or eukaryotic cell membrane. Other constructions may be
used to join nucleic acid sequences encoding HGPRBMY6 protein to
nucleotide sequence encoding a polypeptide domain, which will
facilitate purification of soluble proteins. Such purification
facilitating domains include, but are not limited to, metal
chelating peptides such as histidine-tryptophan modules that allow
purification on immobilized metals; protein A domains that allow
purification on immobilized immunoglobulin; and the domain utilized
in the FLAGS extension/affinity purification system (Immunex Corp.,
Seattle, Wash.). The inclusion of cleavable linker sequences such
as those specific for Factor XA or enterokinase (Invitrogen, San
Diego, Calif.) between the purification domain and HGPRBMY6 protein
may be used to facilitate purification. One such expression vector
provides for expression of a fusion protein containing HGPRBMY6 and
a nucleic acid encoding 6 histidine residues preceding a
thioredoxin or an enterokinase cleavage site. The histidine
residues facilitate purification on IMAC (immobilized metal ion
affinity chromatography) as described by J. Porath et al., 1992,
Prot. Exp. Purif., 3:263-281, while the enterokinase cleavage site
provides a means for purifying from the fusion protein. For a
discussion of suitable vectors for fusion protein production, see
D. J. Kroll et al., 1993; DNA Cell Biol., 12:441-453.
[0132] In addition to recombinant production, fragments of HGPRBMY6
polypeptide may be produced by direct peptide synthesis using
solid-phase techniques (J. Merrifield, 1963, J. Am. Chem. Soc.,
85:2149-2154). Protein synthesis may be performed using manual
techniques or by automation. Automated synthesis may be achieved,
for example, using ABI 431A Peptide Synthesizer (PE Biosystems).
Various fragments of HGPRBMY6 polypeptide can be chemically
synthesized separately and then combined using chemical methods to
produce the fall length molecule.
[0133] Human artificial chromosomes (HACs) may be used to deliver
larger fragments of DNA than can be contained and expressed in a
plasmid vector. HACs are linear microchromosomes which may contain
DNA sequences of 10K to 10M in size, and contain all of the
elements that are required for stable mitotic chromosome
segregation and maintenance (see, J. J. Harrington et al., 1997,
Nature Genet., 15:345-355). HACs of 6 to 10M are constructed and
delivered via conventional delivery methods (e.g., liposomes,
polycationic amino polymers, or vesicles) for therapeutic
purposes.
[0134] Diagnostic Assays
[0135] A variety of protocols for detecting and measuring the
expression of HGPRBMY6 polypeptide using either polyclonal or
monoclonal antibodies specific for the protein are known and
practiced in the art. Examples include enzyme-linked immunosorbent
assay (ELISA), radioimmunoassay (RIA), and fluorescence activated
cell sorting (FACS). A two-site, monoclonal-based immunoassay
utilizing monoclonal antibodies reactive with two non-interfering
epitopes on the HGPRBMY6 polypeptide is preferred, but a
competitive binding assay may also be employed. These and other
assays are described in the art as represented by the publication
of R. Hampton et al., 1990; Serological Methods, a Laboratory
Manual, APS Press, St Paul, Minn. and D. E. Maddox et al., 1983; J.
Exp. Med., 158:1211-1216).
[0136] This invention also relates to the use of HGPRBMY6
polynucleotides as diagnostic reagents. Detection of a mutated form
of the HGPRBMY6 gene associated with a dysfunction will provide a
diagnositc tool that can add to or define a diagnosis of a disease
or susceptibility to a disease which results from under-expression,
over-expression, or altered expression of HGPRBMY6. Individuals
carrying mutations in the HGPRBMY6 gene may be detected at the DNA
level by a variety of techniques.
[0137] Nucleic acids for diagnosis may be obtained from a subject's
cells, such as from blood, urine, saliva, tissue biopsy or autopsy
material. The genomic DNA may be used directly for detection or may
be amplified enzymatically by using PCR or other amplification
techniques prior to analysis. RNA or cDNA may also be used in
similar fashion. Deletions and insertions can be detected by a
change in size of the amplified product in comparison to the normal
genotype. Hybridizing amplified DNA to labeled HGPRBMY6
polynucleotide sequences can identify point mutations. Perfectly
matched sequences can be distinguished from mismatched duplexes by
RNase digestion or by differences in melting temperatures. DNA
sequence differences may also be detected by alterations in
electrophoretic mobility of DNA fragments in gels, with or without
denaturing agents, or by direct DNA sequencing (see, e.g., Myers et
al., Science (1985) 230:1242). Sequence changes at specific
locations may also be revealed by nuclease protection assays, such
as RNase and S1 protection or the chemical cleavage method. See
Cotton et al., Proc. Natl. Acad. Sci., USA (1985) 85:43297-4401. In
another embodiment, an array of oligonucleotides probes comprising
HGPRBMY6 nucleotide sequence or fragments thereof can be
constructed to conduct efficient screening of e.g., genetic
mutations. Array technology methods are well known and have general
applicability and can be used to address a variety of questions in
molecular genetics including gene expression, genetic linkage, and
genetic variability (see for example: M. Chee et al., Science,
274:610-613, 1996).
[0138] The diagnostic assays offer a process for diagnosing or
determining a susceptibility to infections such as bacterial,
fungal, protozoan and viral infections, particularly infections
caused by HIV-1 or HIV-2 through detection of a mutation in the
HGPRBMY6 gene by the methods described. The invention also provides
diagnostic assays for determining or monitoring susceptibility to
the following conditions, diseses, or disorders: cancers; anorexia;
bulimia asthma; Parkinson's disease; acute heart failure;
hypotension; hypertension; urinary retention; osteoporosis; angina
pectoris; myocardial infarction; ulcers; asthma; allergies; benign
prostatic hypertrophy; and psychotic and neurological disorders,
including anxiety, schizophrenia, manic depression, delirium,
dementia, severe mental retardation and dyskinesias, such as
Huntington's disease or Gilles dela Tourett's syndrome.
[0139] In addition, infections such as bacterial, protozoan and
viral infections, particularly infections caused by HIV-1 or HIV-2;
as well as, conditions or disorders such as pain; cancers;
anorexia; bulimia; asthma; Parkinson's disease; acute heart
failure; hypotension; hypertension; urinary retention;
osteoporosis; angina pectoris; myocardial infarction; ulcers;
asthma; allergies; benign prostatic hypertrophy; and psychotic and
neurological disorders, including anxiety, schizophrenia, manic
depression, delirium, dementia, severe mental retardation and
dyskinesias, such as Huntington's disease or Gilles dela Tourett's
syndrome, can be diagnosed by methods comprising determining from a
sample derived from a subject having an abnormally decreased or
increased level of HGPRBMY6 polypeptide or HGPRBMY6 mRNA. Decreased
or increased expression can be measured at the RNA level using any
of the methods well known in the art for the quantificatoin of
polynucleotides, such as, for example, PCR, RT-PCR, RNase
protection, Northern blotting and other hybridization methods.
Assay techniques that can be used to determine levels of a protein,
such as an HGPRBMY6, in a sample derived from a host are well known
to those of skill in the art. Such assay methods include
radioimmunoassays, competitive-binding assays, Western Blot
analysis, and ELISA assays.
[0140] In another of its aspects, the present invention relates to
a diagnostic kit for a disease or susceptibility to a disease,
particularly infections such as bacterial, fungal, protozoan and
viral infections, particularly infections caused by HIV-1 or HIV-2;
pain; cancers; anorexia; bulimia; asthma; Parkinson's disease;
acute heart failure; hypotension; hypertension; urinary retention;
osteoporosis; angina pectoris; myocardial infarction; ulcers;
asthma; allergies; benign prostatic hypertrophy, and psychotic and
neurological disorders, including anxiety, schizophrenia, manic
depression, delirium, dementia, severe medal retardation and
dyskinesias, such as Huntington's disease or Gilles dela Tourett's
syndrome, which comprises:
[0141] (a) a HGPRBMY6 polynucleotide, preferably the nucleotide
sequence of SEQ ID NO: 1, or a fragment thereof; or
[0142] (b) a nucleotide sequence complementary to that of (a);
or
[0143] (c) a HGPRBMY6 polypeptide, preferably the polypeptide of
SEQ ID NO: 2, or a fragment thereof; or
[0144] (d) an antibody to a HGPRBMY6 polypeptide, preferably to the
polypeptide of SEQ ID NO: 2, or combinations thereof.
[0145] It will be appreciated that in any such kit, (a), (b), (c)
or (d) may comprise a substantial component.
[0146] The GPCR polynucleotides which may be used in the diagnostic
assays according to the present invention include oligonucleotide
sequences, complementary RNA and DNA molecules, and PNAs. The
polynucleotides may be used to detect and quantify
HGPRBMY6-encoding nucleic acid expression in biopsied tissues in
which expression (or under- or overexpression) of the HGPRBMY6
polynucleotide may be correlated with disease. The diagnostic
assays may be used to distinguish between the absence, presence,
and excess expression of HGPRBMY6, and to monitor regulation of
HGPRBMY6 polynucleotide levels during therapeutic treatment or
intervention.
[0147] In a related aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding HGPRBMY6 polypeptide, or closely related
molecules, may be used to identify nucleic acid sequences which
encode HGPRBMY6 polypeptide. The specificity of the probe, whether
it is made from a highly specific region, e.g., about 8 to 10
contiguous nucleotides in the 5' regulatory region, or a less
specific region, e.g., especially in the 3' coding region, and the
stringency of the hybridization or amplification (maximal, high,
intermediate, or low) will determine whether the probe identifies
only naturally occurring sequences encoding HGPRBMY6 polypeptide,
alleles thereof, or related sequences.
[0148] Probes may also be used for the detection of related
sequences, and should preferably contain at least 50% of the
nucleotides encoding the HGPRBMY6 polypeptide. The hybridization
probes of this invention may be DNA or RNA and may be derived from
the nucleotide sequence of SEQ ID NO:1, or from genomic sequence
including promoter, enhancer elements, and introns of the naturally
occurring HGPRBMY6 protein.
[0149] Methods for producing specific hybridization probes for DNA
encoding the HGPRBMY6 polypeptide include the cloning of a nucleic
acid sequence that encodes the HGPRBMY6 polypeptide, or HGPRBMY6
derivatives, into vectors for the production of mRNA probes. Such
vectors are known in the art, commercially available, and may be
used to synthesize RNA probes in vitro by means of the addition of
the appropriate RNA polymerases and the appropriate labeled
nucleotides. Hybridization probes may be labeled by a variety of
detector/reporter groups, e.g., radionuclides such as .sup.32P or
.sup.35S, or enzymatic labels, such as alkaline phosphatase coupled
to the probe via avidin/biotin coupling systems, and the like.
[0150] The polynucleotide sequence encoding the HGPRBMY6
polypeptide, or fragments thereof, may be used for the diagnosis of
disorders associated with expression of HGPRBMY6. Examples of such
disorders or conditions are described above for "Therapeutics". The
polynucleotide sequence encoding the HGPRBMY6 polypeptide may be
used in Southern or Northern analysis, dot blot, or other
membrane-based technologies; in PCR technologies; or in dip stick,
pin, ELISA or chip assays utilizing fluids or tissues from patient
biopsies to detect the status of, e.g., levels or overexpression of
HGPRBMY6, or to detect altered HGPRBMY6 expression. Such
qualitative or quantitative methods are well known in the art.
[0151] In a particular aspect, the nucleotide sequence encoding the
HGPRBMY6 polypeptide may be useful in assays that detect activation
or induction of various neoplasms or cancers, particularly those
mentioned supra. The nucleotide sequence encoding the HGPRBMY6
polypeptide may be labeled by standard methods, and added to a
fluid or tissue sample from a patient, under conditions suitable
for the formation of hybridization complexes. After a suitable
incubation period, the sample is washed and the signal is
quantified and compared with a standard value. If the amount of
signal in the biopsied or extracted sample is significantly altered
from that of a comparable control sample, the nucleotide sequence
has hybridized with nucleotide sequence present in the sample, and
the presence of altered levels of nucleotide sequence encoding the
HGPRBMY6 polypeptide in the sample indicates the presence of the
associated disease. Such assays may also be used to evaluate the
efficacy of a particular therapeutic treatment regimen in animal
studies, in clinical trials, or in monitoring the treatment of an
individual patient.
[0152] To provide a basis for the diagnosis of disease associated
with expression of HGPRBMY6, a normal or standard profile for
expression is established. This may be accomplished by combining
body fluids or cell extracts taken from normal subjects, either
animal or human, with a sequence, or a fragment thereof, which
encodes the HGPRBMY6 polypeptide, under conditions suitable for
hybridization or amplification. Standard hybridization may be
quantified by comparing the values obtained from normal subjects
with those from an experiment where a known amount of a
substantially purified polynucleotide is used. Standard values
obtained from normal samples may be compared with values obtained
from samples from patients who are symptomatic for disease.
Deviation between standard and subject (patient) values is used to
establish the presence of disease.
[0153] Once disease is established and a treatment protocol is
initiated, hybridization assays may be repeated on a regular basis
to evaluate whether the level of expression in the patient begins
to approximate that which is observed in a normal individual. The
results obtained from successive assays may be used to show the
efficacy of treatment over a period ranging from several days to
months.
[0154] With respect to cancer, the presence of an abnormal amount
of transcript in biopsied tissue from an individual may indicate a
predisposition for the development of the disease, or may provide a
means for detecting the disease prior to the appearance of actual
clinical symptoms. A more definitive diagnosis of this type may
allow health professionals to employ preventative measures or
aggressive treatment earlier, thereby preventing the development or
further progression of the cancer.
[0155] Additional diagnostic uses for oligonucleotides designed
from the nucleic acid sequence encoding the HGPRBMY6 polypeptide
may involve the use of PCR. Such oligomers may be chemically
synthesized, generated enzymatically, or produced from a
recombinant source. Oligomers will preferably comprise two
nucleotide sequences, one with sense orientation (5'.fwdarw.3') and
another with antisense (3'.fwdarw.5'), employed under optimized
conditions for identification of a specific gene or condition. The
same two oligomers, nested sets of oligomers, or even a degenerate
pool of oligomers may be employed under less stringent conditions
for detection and/or quantification of closely related DNA or RNA
sequences.
[0156] Methods suitable for quantifying the expression of HGPRBMY6
include radiolabeling or biotinylating nueleotides,
co-amplification of a control nucleic acid, and standard curves
onto which the experimental results are interpolated (P. C. Melby
et al., 1993, J. Immunol. Methods, 159:235-244; and C. Duplaa et
al., 1993, Anal. Biochem., 229-236). The speed of quantifying
multiple samples may be accelerated by running the assay in an
ELISA format where the oligomer of interest is presented in various
dilutions and a spectrophotometric or colorimetric response gives
rapid quantification.
[0157] Therapeutic Assays
[0158] HGPRBMY6 polypeptide shares homology with G-protein coupled
receptors, more specifically, latrophilin, alphalatrotoxin and CL3
receptors. Because HGPRBMY6 is highly expressed in small intestine
and colonic tissue, the HGPRBMY6 product may play a role in
gastrointestinal disorders, and/or in cell cycle regulation, and/or
in cell signaling. The HGPRBMY6 protein may be further involved in
neoplastic, gastrointestinal, and neurological disorders.
[0159] In one embodiment of the present invention, the HGPRBMY6
protein may play a role in neoplastic disorders. An antagonist of
HGPRBMY6 polypeptide may be administered to an individual to
prevent or treat a neoplastic disorder. Such disorders may include,
but are not limited to, adenocarcinoma, leukemia, lymphoma,
melanoma, myeloma, sarcoma, and teratocarcinoma, and particularly,
cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall bladder, ganglia, gastrointestinal tract,
heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid,
penis, prostate, salivary
[0160] In a related aspect, an antibody which specifically binds to
HGPRBMY6 may be used directly as an antagonist or indirectly as a
targeting or delivery mechanism for bringing a pharmaceutical agent
to cells or tissue which express HGPRBMY6 polypeptide.
[0161] In another embodiment of the present invention, an
antagonist or inhibitory agent of the HGPRBMY6 polypeptide may be
administered to a subject to prevent or treat a neurological
disorder. Such disorders may include, but are not limited to,
akathesia, Alzheimer's disease, amnesia, amyotrophic lateral
sclerosis, bipolar disorder, catatonia, cerebral neoplasms,
dementia, depression, Down's syndrome, tardive dyskinesia,
dystonias, epilepsy, Huntington's disease, multiple sclerosis,
Parkinson's disease, paranoid psychoses, schizophrenia, and
Tourette's disorder.
[0162] In another embodiment of the present invention, an
antagonist or inhibitory agent of the HGPRBMY6 polypeptide may be
administered to an individual to prevent or treat an immunological
disorder. Such disorders may include, but are not limited to, AIDS,
Addison's disease, adult respiratory distress syndrome, allergies,
anemia, asthma, atherosclerosis, bronchitis, cholecystitis, Crohn's
disease, ulcerative colitis, atopic dermatitis, dermatomyositis,
diabetes mellitus, emphysema, erythema nodosum, atrophic gastritis,
glomerulonephritis, gout, Graves'disease, hypereosinophilia,
irritable bowel syndrome, lupus erythematosus, multiple sclerosis,
myasthenia gravis, myocardial or pericardial inflammation,
osteoarthritis, osteoporosis, pancreatitis, polymyositis,
rheumatoid arthritis, scleroderma, Sjogren's syndrome, and
autoimmune thyroiditis; complications of cancer, hemodialysis,
extracorporeal circulation; viral, bacterial, fungal, parasitic,
protozoal, and helminthic infections and trauma.
[0163] In a preferred embodiment of the present invention, an
antagonist or inhibitory agent of the HGPRBMY6 polypeptide may be
administered to an individual to prevent or treat a small
intestine- or colon-related disorder, particularly since HGPRBMY6
is highly expressed in the small intestine and colon. Such
conditions or disorders may include, but are not limited to,
intestinal bowel disorder, ulceritis, ulceritis colitis, Crohn's
disease, colon cancer, psoriasis, angiodysplasias, and gastric
heterotopia.
[0164] In preferred embodiments, the HGPRBMY6 polynucleotides and
polypeptides, including agonists, antagonists, and fragments
thereof, are useful for modulating intracellular cAMP associated
signaling pathways.
[0165] In preferred embodiments, the HGPRBMY6 polynucleotides and
polypeptides, including agonists, antagonists, and fragments
thereof, are useful for modulating intracellular Ca.sup.2+ levels,
modulating Ca.sup.2+ sensitive signaling pathways via G alpha 15,
and modulating NFAT element associated signaling pathways.
[0166] In another embodiment of the present invention, an
expression vector containing the complement of the polynucleotide
encoding HGPRBMY6 polypeptide may be administered to an individual
to treat or prevent a neoplastic disorder, including, but not
limited to, the types of cancers and tumors described above.
[0167] In another embodiment of the present invention, an
expression vector containing the complement of the polynucleotide
encoding the HGPRBMY6 polypeptide may be administered to an
individual to treat or prevent a neurological disorder, including,
but not limited to, the types of disorders described above.
[0168] In yet another embodiment of the present invention, an
expression vector containing the complement of the polynucleotide
encoding the HGPRBMY6 polypeptide may be administered to an
individual to treat or prevent an gastrointestinal disorder,
including, but not limited to, the types of small intestine- or
colon-related disorders described above.
[0169] In another embodiment, the proteins, antagonists,
antibodies, agonists, complementary sequences, or vectors of the
present invention can be administered in combination with other
appropriate therapeutic agents. Selection of the appropriate agents
for use in combination therapy may be made by one of ordinary skill
in the art, according to conventional pharmaceutical principles.
The combination of therapeutic agents may act synergistically to
effect the treatment or prevention of the various disorders
described above. Using this approach, one may be able to achieve
therapeutic efficacy with lower dosages of each agent, thus
reducing the potential for adverse side effects.
[0170] Antagonists or inhibitors of the HGPRBMY6 polypeptide (SEQ
ID NO:2) of the present invention may be produced using methods
which are generally known in the art. For example, the HGPRBMY6
transfected CHO-NFAT/CRE cell lines of the present invention are
useful for the identification of agonists and antagonists of the
HGPRBMY6 polypeptide. Representative uses of these cell lines would
be their inclusion in a method of identifying HGPRBMY6 agonists and
antagonists. Preferably, the cell lines are useful in a method for
identifying a compound that modulates the biological activity of
the HGPRBMY6 polypeptide, comprising the steps of (a) combining a
candidate modulator compound with a host cell expressing the
HGPRBMY6 polypeptide having the sequence as set forth in SEQ ID
NO:2; and (b) measuring an effect of the candidate modulator
compound on the activity of the expressed HGPRBMY6 polypeptide.
Representative vectors expressing the HGPRBMY6 polypeptide are
referenced herein (e.g., pcDNA3.1 hygro.TM.) or otherwise known in
the art.
[0171] The cell lines are also useful in a method of screening for
a compounds that is capable of modulating the biological activity
of HGPRBMY6 polypeptide, comprising the steps of: (a) determining
the biological activity of the HGPRBMY6 polypeptide in the absence
of a modulator compound; (b) contacting a host cell expression the
HGPRBMY6 polypeptide with the modulator compound; and (c)
determining the biological activity of the HGPRBMY6 polypeptide in
the presence of the modulator compound; wherein a difference
between the activity of the HGPRBMY6 polypeptide in the presence of
the modulator compound and in the absence of the modulator compound
indicates a modulating effect of the compound. Additional uses for
these cell lines are described herein or otherwise known in the
art. In particular, purified HGPRBMY6 protein, or fragments
thereof, can be used to produce antibodies, or to screen libraries
of pharmaceutical agents, to identify those which specifically bind
HGPRBMY6.
[0172] Antibodies specific for HGPRBMY6 polypeptide, or immunogenic
peptide fragments thereof, can be generated using methods that have
long been known and conventionally practiced in the art. Such
antibodies may include, but are not limited to, polyclonal,
monoclonal, chimeric, single chain, Fab fragments, and fragments
produced by an Fab expression library. Neutralizing antibodies,
(i.e., those which inhibit dimer formation) are especially
preferred for therapeutic use.
[0173] The present invention also encompasses the polypeptide
sequences that intervene between each of the predicted HGPRBMY6
transmembrane domains. Since these regions are solvent accessible
either extracellularly or intracellularly, they are particularly
useful for designing antibodies specific to each region. Such
antibodies may be useful as antagonists or agonists of the HGPRBMY6
full-length polypeptide and may modulate its activity.
[0174] The following serve as non-limiting examples of peptides or
fragments that may be used to generate antibodies:
[0175] METYSLSLGNQSVVEPNIAIQSANFSSENAVGPSNVRFSVQKGASSSLVSSSTFI
HTNVDGLNPDAQTELQVLLNMTKNYTKTCGFVVYQNDKLFQSKTFTAKS
DFSQKIISSKTDENEQDQSASVD- MVFSPKYNQKEFQLYSYACVYWNLSAK
DWDTYGCQKDKGTDGFLRCRCNHTTNFAVLMTFKKDYQYPKSLD (SEQ ID NO:12)
[0176] QIVTRKVRKT (SEQ ID NO:13)
[0177] ENSNKNLQTSDGDINNIDFDNNDIPRTDTINIPNPMCT (SEQ ID NO:14)
[0178] IRTMKPLPRH (SEQ ID NO:15)
[0179] TVGVIYSQNGNNPQWELDYRQEKICWLAIPEPNGVIKSPLL (SEQ ID NO:16)
[0180] TISIKVLWKNNQNLTSTKKVSSMKK (SEQ ID NO:17)
[0181] NDDSIR (SEQ ID NO:18)
[0182] YTVRTKVFQSEASKVLMLLSSIGRRKSLPSVTRPRLRVKMYNFLRSLPTLHERF
RLLETSPSTEEITLSESDNAKESI (SEQ ID NO:19)
[0183] In preferred embodiments, the following N-terminal HGPRBMY6
N-terminal fragment deletion polypeptides are encompassed by the
present invention: M1-D198, E2-D198, T3-D198, Y4-D198, S5-D198,
L6-D198, S7-D198, L8-d198, G9-D198, N10-D198, Q11-D198, S12-D198,
V13-D198, V14-D198, E15-D198, P16-D198, N17-D198, I18-D198,
A19-D198, I20-D198, Q21-D198, S22-D198, A23-D198, N24-D198,
F25-D198, S26-D198, S27-D198, E28-D198, N29-D198, A30-D198,
V31-D198, G32-D198, P33-D198, S34-D198, N35-D198, V36-D198,
R37-D198, F38-D198, S39-D198, V40-D198, Q41-D198, K42-D198,
G43-D198, A44-D198, S45-D198, S46-D198, S47-D198, L48-D198,
V49-D198, S50-D198, S51-D198, S52-D198, T53-D198, F54-D198,
I155-D198, H56-D198, T57-D198, N58-D198, V59-D198, D60-D198,
G61-D198, L62-D198, N63-D198, P64-D198, D65-D198, A66-D198,
Q67-D198, T68-D198, E69-D198, L70-D198, Q71-D198, V72-D198,
L73-D198, L74-D198, N75-D198, M76-D198, T77-D198, K78-D198,
N79-D198, Y80-D198, T81-D198, K82-D198, T83-D198, C84-D198,
G85-D198, F86-D198, V87-D198, V88-D198, Y89-D198, Q90-D198,
N91-D198, D92-D198, K93-D198, L94-D198, F95-D198, Q96-D198,
S97-D198, K98-D198, T99-D198, F100-D198, T101-D198, A102-D198,
K103-D198, S104-D198, D105-D198, F106-D198, S107-D198, Q108-D198,
K109-D198, I111-D198, S112-D198, S112-D198, S113-D198, K114-D198,
T115-D198, D116-D198, E117-D198, N118-D198, E119-D198, Q120-D198,
D121-D198, Q122-D198, S123-D198, A124-D198, S125-D198, V126-D198,
D127-D198, M128-D198, V129-D198, F130-D198, S131-D198, P132-D198,
K133-D198, Y134-D198, N135-D198, Q136-D198, K137-D198, E138-D198,
F139-D198, Q140-D198, L141-D198, Y142-D198, S143-D198, Y144-D198,
A145-D198, C146-D198, V147-D198, Y148-D198, W149-D198, N150-D198,
L151-D198, S152-D198, A153-D198, K154-D198, D155-D198, W156-D198,
D157-D198, T158-D198, Y159-D198, G160-D198, C161-D198, Q162-D198,
K163-D198, D164-D198, K165-D198, G166-D198, T167-D198, D168-D198,
G169-D198, F170-D198, L171-D198, R172-D198, C173-D198, R174-D198,
C175-D198, N176-D198, H177-D198, T178-D198, T179-D198, N180-D198,
F181-D198, A182-D198, V183-D198, L184-D198, M185-D198, T186-D198,
F187-D198, K188-D198, K189-D198, D190-D198, Y191-D198, and/or
Q192-D198 of SEQ ID NO:12. Polynucleotide sequences encoding these
polypeptides are also provided. The present invention also
encompasses the use of these N-terminal HGPRBMY6 N-terminal
fragment deletion polypeptides as immunogenic and/or antigenic
epitopes as described elsewhere herein.
[0184] In preferred embodiments, the following C-terminal HGPRBMY6
N-terminal fragment deletion polypeptides are encompassed by the
present invention: M1-D198, M1-L197, M1-S196, M1-K195, M1-P194,
M1-Y193, M 1-Q192, M1-Y191, M1-D190, M1-K189, M1-K188, M1-F187,
M1-T186, M1-M185, M1-L184, M1-V183, M1-A182, M1-F181, M1-N180,
M1-T179, M1-T178, M1-H177, M1-N176, M1-C175, M1-R174, M1-C173,
M1-R172, M1-L171, M1-F170, M1-G169, M1-D168, M1-T167, M1-G166,
M1-K165, M1-D164, M1-K163, M1-Q162, M1-C161, M1-G160, M1-Y159,
M1-T158, M1-D157, M1-W156, M1-D155, M1-K154, M1-A153, M1-S152,
M1-L151, M1-N150, M1-W149, M1-Y148, M1-V147, M1-C146, M1-A145,
M1-Y144, M1-S143, M1-Y142, M1-L141, M1-Q140, M1-F139, M1-E138,
M1-K137, M1-Q136, M1-N135, M1-Y134, M1-K133, M1-P132, M1-S131,
M1-F130, M1-V129, M1-M128, M1-D127, M1-V126, M1-S125, M1-A124,
M1-S123, M1-Q122, M1-D121, M1-Q120, M1-E119, M1-N118, M1-E117,
M1-D116, M1-T115, M1-K114, M1-S113, M1-S112, M1-I111, M1-I110,
M1-K109, M1-Q108, M1-S107, M1-F106, M1-D105, M1-S104, M1-K103,
M1-A102, M1-T101, M1-F100, M1-T99, M1-K98, M1-S97, M1-Q96, M1-F95,
M1-L94, M1-K93, M1-D92, M1-N91, M1-Q90, M1-Y89, M1-V88, M1-V87,
M1-F86, M1-G85, M1-C84, M1-T83, M1-K82, M1-T81, M1-Y80, M1-N79,
M1-K78, M1-T77, M1-M76, M1-N75, M1-L74, M1-L73, M1-V72, M1-Q71,
M1-L70, M1-E69, M1-T68, M1-Q67, M1-A66, M1-D65, M1-P64, M1-N63,
M1-L62, M1-G61, M1-D60, M1-V59, M1-N58, M1-T57, M1-H56, M1-I55,
M1-F54, M1-T53, M1-S52, , M1-S51, M1-S50, M1-V49, M1-L48, M1-S47,
M1-S46, M1-S45, M1-A44, M1-G43, M1-K42, M1-Q41, M1-V40, M1-S39,
M1-F38, M1-R37, M1-V36, M1-N35, M1-S34, M1-P33, M1-G32, M1-V31,
M1-A30, M1-N29, M1-E28, M1-S27, M1-S26, M1-F25, M1-N24, M1-A23,
M1-S22, M1-Q21, M1-I20, M1-A19, M1-I18, M1-N17, M1-P16, M1-E15,
M1-V14, M1-V13, M1-S12, M1-Q11, M1-N10, M1-G9, M1-L8, and/or M1-S7
of SEQ ID NO:12. Polynucleotide sequences encoding these
polypeptides are also provided. The present invention also
encompasses the use of these C-terminal HGPRBMY6 N-terninal
fragment deletion polypeptides as immunogenic and/or antigenic
epitopes as described elsewhere herein.
[0185] In preferred embodiments, the following N-terminal HGPRBMY6
TM1-2 intertransmembrane domain deletion polypeptides are
encompassed by the present invention: Q1-T10, I2-T10, V3-T10,
and/or T4-T10 of SEQ ID NO:13. Polynucleotide sequences encoding
these polypeptides are also provided. The present invention also
encompasses the use of these N-terminal HGPRBMY6 TM1-2
intertransmembrane domain deletion polypeptides as immunogenic
and/or antigenic epitopes as described elsewhere herein.
[0186] In preferred embodiments, the following C-terminal HGPRBMY6
TM1-2 intertransmembrane domain deletion polypeptides are
encompassed by the present invention: Q1-T10, Q1-K9, Q1-R8, and/or
Q1-V7 of SEQ ID NO:13. Polynucleotide sequences encoding these
polypeptides are also provided. The present invention also
encompasses the use of these C-terminal HGPRBMY6 TM1-2
intertransmembrane domain deletion polypeptides as immunogenic
and/or antigenic epitopes as described elsewhere herein.
[0187] In preferred embodiments, the following N-terminal HGPRBMY6
TM2-3 intertransmembrane domain deletion polypeptides are
encompassed by the present invention: E1-T38, N2-T38, S3-T38,
N4-T38, K5-T38, N6-T38, L7-T38, Q8-T38, T9-T38, S10-T38, D11-T38,
G12-T38, D13-T38, I14-T38, N15-T38, N16-T38, I17-T38, D18-T38,
F19-T38, D20-T38, N21-T38, N22-T38, D23-T38, I24-T38, P25-T38,
R26-T38, T27-T38, D28-T38, T29-T38, I30-T38, N31-T38, and/or
I32-T38 of SEQ ID NO:14. Polynucleotide sequences encoding these
polypeptides are also provided. The present invention also
encompasses the use of these N-terminal HGPRBMY6 TM2-3
intertransmembrane domain deletion polypeptides as immunogenic
and/or antigenic epitopes as described elsewhere herein.
[0188] In preferred embodiments, the following C-terminal HGPRBMY6
TM2-3 intertransmembrane domain deletion polypeptides are
encompassed by the present invention: E1-T38, E1-C37, E1-M36,
E1-P35, E1-N34, E1-P33, E1-132, E1-N31, E1-30, E1-T29, E1-D28,
E1-T27, E1-R26, E1-P25, E1-I24, E1-N22, E1-N21, E1-D20, E1-F19,
E1-D18, E1-I17, E1-N16, E1-N15, E1-I14, E1-D13, E1-G12, E1-D11,
E1-S10, E1-T9, E1-Q8, and/or E1-L7 of SEQ ID NO:14. Polynucleotide
sequences encoding these polypeptides are also provided. The
present invention also encompasses the use of these C-terminal
HGPRBMY6 TM2-3 intertransmembrane domain deletion polypeptides as
immunogenic and/or antigenic epitopes as described elsewhere
herein.
[0189] In preferred embodiments, the following N-terminal HGPRBMY6
TM3-4 intertransmembrane domain deletion polypeptides are
encompassed by the present invention: I1-H10, R2-H10, T3-H10,
and/or M4-H10 of SEQ ID NO:15. Polynucleotide sequences encoding
these polypeptides are also provided. The present invention also
encompasses the use of these N-terminal HGPRBMY6 TM3-4
intertransmembrane domain deletion polypeptides as immunogenic
and/or antigenic epitopes as described elsewhere herein.
[0190] In preferred embodiments, the following C-terminal HGPRBMY6
TM3-4 intertransmembrane domain deletion polypeptides are
encompassed by the present invention: I1-H10, I1-R9, I1-P8, and/or
I1-L7 of SEQ ID NO:15. Polynucleotide sequences encoding these
polypeptides are also provided. The present invention also
encompasses the use of these C-terminal HGPRBMY6 TM3-4
intertransmembrane domain deletion polypeptides as immunogenic
and/or antigenic epitopes as described elsewhere herein.
[0191] In preferred embodiments, the following N-terminal HGPRBMY6
TM4-5 intertransmembrane domain deletion polypeptides are
encompassed by the present invention: T1-L41, V2-L41, G3-L41,
V4-L41, I5-L41, Y6-L41, S7-L41, Q8-L41, N9-L41, G10-L41, N11-L41,
N12-L41, P13-L41, Q14-L41, W15-L41, E16-L41, L17-L41, D18-L41,
Y19-L41, R20-L41, Q21-L41, E22-L41, K23-L41, I24-L41, C25-L41,
W26-L41, L27-L41, A28-L41, I29-L41, P30-L41, E31-L41, P32-L41,
N33-L41, G34-L41, and/or V35-L41 of SEQ ID NO:16. Polynucleotide
sequences encoding these polypeptides are also provided. The
present invention also encompasses the use of these N-terminal
HGPRBMY6 TM4-5 intertransmembrane domain deletion polypeptides as
immunogenic and/or antigenic epitopes as described elsewhere
herein.
[0192] In preferred embodiments, the following C-terminal HGPRBMY6
TM4-5 intertransmembrane domain deletion polypeptides are
encompassed by the present invention: T1-L41, T1-L40, T1-P39,
T1-S38, T1-K37, T1-I36, T1-V35, T1-G34, T1-N33, T1-P32, T1-E31,
T1-P30, T1-129, T1-A28, T1-L27, T1-W26, T1-C25, T1-I24, T1-K23,
T1-E22, T1-Q21, T1-R20, T1-Y19, T1-D18, T1-L17, T1-E16, T1-W15,
T1-Q14, T1-P13, T1-N12, T1-N11, T1-G10, T1-N9, T1-Q8, and/or T1-S7
of SEQ ID NO:16. Polynucleotide sequences encoding these
polypeptides are also provided. The present invention also
encompasses the use of these C-terminal HGPRBMY6 TM4-5
intertransmembrane domain deletion polypeptides as immunogenic
and/or antigenic epitopes as described elsewhere herein.
[0193] In preferred embodiments, the following N-terminal HGPRBMY6
TM5-6 intertransmembrane domain deletion polypeptides are
encompassed by the present invention: T1-K25, I2-K25, S3-K25,
I4-K25, K5-K25, V6-K25, L7-K25, W8-K25, K9-K25, N10-K25, N11-K25,
Q12-K25, N13-K25, L14-K25, T15-K25, S16-K25, T17-K25, K18-K25,
and/or K19-K25 of SEQ ID NO:16. Polynucleotide sequences encoding
these polypeptides are also provided. The present invention also
encompasses the use of these N-terminal HGPRBMY6 TM5-6
intertransmembrane domain deletion polypeptides as immunogenic
and/or antigenic epitopes as described elsewhere herein.
[0194] In preferred embodiments, the following C-terminal HGPRBMY6
TM5-6 intertransmembrane domain deletion polypeptides are
encompassed by the present invention: T1-K25, T1-K24, T1-M23,
T1-S22, T1-S21, T1-V20, T1-K19, T1-K18, T1-T17, T1-S16, T1-T15,
T1-L14, T1-N13, T1-Q12, T1-N11, T1-N10, T1-K9, T1-W8, and/or T1-L7
of SEQ ID NO:16. Polynucleotide sequences encoding these
polypeptides are also provided. The present invention also
encompasses the use of these C-terminal HGPRBMY6 TM5-6
intertransmembrane domain deletion polypeptides as immunogenic
and/or antigenic epitopes as described elsewhere herein.
[0195] In preferred embodiments, the following N-terminal HGPRBMY6
C-terminal fragment deletion polypeptides are encompassed by the
present invention: Y1-I78, T2-I78, V3-I78, R4-I78, T5-I78, K6-I78,
V7-I78, F8-I78, Q9-I78, S10-I78, E11-I78, A12-I78, S13-I78,
K14-I78, V15-I78, L16-I78, M17-I78, L18-I78, L19-I78, S20-I78,
S21-I78, I22-I78, G23-I78, R24-I78, R25-I78, K26-I78, S27-I78,
L28-I78, P29-I78, S30-I78, V31-I78, T32-I78, R33-I78, P34-I78,
R35-I78, L36-I78, R37-I78, V38-I78, K39-I78, M40-I78, Y41-I78,
N42-I78, F43-I78, L44-I78, R45-I78, S46-I78, L47-I78, P48-I78,
T49-I78, L50-I78, H51-I78, E52-I78, R53-I78, F54-I78, R55-I78,
L56-I78, L57-I78, E58-I78, T59-I78, S60-I78, S62-I78, T63-I78,
E64-I78, E65-I78, I66-I78, T67-I78, L68-I78, S69-I78, E70-I78,
S71-I78, and/or D72-I78 of SEQ ID NO:19. Polynucleotide sequences
encoding these polypeptides are also provided. The present
invention also encompasses the use of these N-terminal HGPRBMY6
C-terminal fragment deletion polypeptides as immunogenic and/or
antigenic epitopes as described elsewhere herein.
[0196] In preferred embodiments, the following C-terminal HGPRBMY6
C-terminal fragment deletion polypeptides are encompassed by the
present invention: Y1-I78, Y1-S77, Y1-E76, Y1-K75, Y1-A74, Y1-N73,
Y1-D72, Y1-S71, Y1-E70, Y1-S69, Y1-L68, Y1-T67, Y1-I66, Y1-E65,
Y1-E64, Y1-T63, Y1-S62, Y1-P61, Y1-S60, Y1-T59, Y1-E58, Y1-L57,
Y1-L56, Y1-R55, Y1-F54, Y1-R53, Y1-E52, Y1-H51, Y1-L50, Y1-T49,
Y1-P48, Y1-L47, Y1-S46, Y1-R45, Y1-L44, Y1-F43, Y1-N42, Y1-Y41,
Y1-M40, Y1-K39, Y1-V38, Y1-R37, Y1-L36, Y1-R353, Y1-P34, Y1-R33,
Y1-T32, Y1-V31, Y1-S30, Y1-P29, Y1-L28, Y1-S27, Y1-K26, Y1-R25,
Y1-R24, Y1-G23, Y1-I22, Y1-S21, Y1-S20, Y1-L19, Y1-L18, Y1-M17,
Y1-L16, Y1-V15, Y1-K14, Y1-S13, Y1-A12, Y1-E11, Y1-S10, Y1-Q9,
Y1-F8, and/or Y1-V7 of SEQ ID NO:19. Polynucleotide sequences
encoding these polypeptides are also provided. The present
invention also encompasses the use of these C-terminal HGPRBMY6
C-terminal fragment deletion polypeptides as immunogenic and/or
antigenic epitopes as described elsewhere herein.
[0197] The HGPRBMY6 polypeptides of the present invention were
determined to comprise several phosphorylation sites based upon the
Motif algorithm (Genetics Computer Group, Inc.). The
phosphorylation of such sites may regulate some biological activity
of the HGPRBMY6 polypeptide. For example, phosphorylation at
specific sites may be involved in regulating the proteins ability
to associate or bind to other molecules (e.g., proteins, ligands,
substrates, DNA, etc.). In the present case, phosphorylation may
modulate the ability of the HGPRBMY6 polypeptide to associate with
other polypeptides, particularly cognate ligand for HGPRBMY6, or
its ability to modulate certain cellular signal pathways.
[0198] The HGPRBMY6 polypeptide was predicted to comprise fifteen
PKC phosphorylation sites using the Motif algorithm (Genetics
Computer Group, Inc.). In vivo, protein kinase C exhibits a
preference for the phosphorylation of serine or threonine residues.
The PKC phosphorylation sites have the following consensus pattern:
[ST]-x-[RK], where S or T represents the site of phosphorylation
and `x` an intervening amino acid residue. Additional information
regarding PKC phosphorylation sites can be found in Woodget J. R.,
Gould K. L., Hunter T., Eur. J. Biochem. 161:177-184(1986), and
Kishimoto A., Nishiyama K., Nakanishi H., Uratsuji Y., Nomura H.,
Takeyama Y., Nishizuka Y., J. Biol. Chem. 260:12492-12499(1985);
which are hereby incorporated by reference herein.
[0199] In preferred embodiments, the following PKC phosphorylation
site polypeptides are encompassed by the present invention:
QSKTFTAKSDFSQ (SEQ ID NO:27), AKSDFSQKIISSK (SEQ ID NO:28),
SQKIISSKTDENE (SEQ ID NO:29), VDMVFSPKYNQKE (SEQ ID NO:30),
VYWNLSAKDWDTY (SEQ ID NO:31), FAVLMTFKKDYQY (SEQ ID NO:32),
IFQIVTRKVRKTS (SEQ ID NO:33), FGIENSNKNLQTS (SEQ ID NO:34),
YLLIRTMKPLPRH (SEQ ID NO:35), MFITISIKVLWKN (SEQ ID NO:36),
NQNLTSTKKVSSM (SEQ ID NO:37), QNLTSTKKVSSMK (SEQ ID NO:38),
TKKVSSMKKIVST (SEQ ID NO:39), LVNDDSIRIVFSY (SEQ ID NO:40), and/or
IFILYTVRTKVFQ (SEQ ID NO:41). Polynucleotides encoding these
polypeptides are also provided. The present invention also
encompasses the use of the HGPRBMY6 PKC phosphorylation site
polypeptides as immunogenic and/or antigenic epitopes as described
elsewhere herein.
[0200] The HGPRBMY6 polypeptide was predicted to comprise nine
casein kinase II phosphorylation sites using the Motif algorithm
(Genetics Computer Group, Inc.). Casein kinase II (CK-2) is a
protein serine/threonine kinase whose activity is independent of
cyclic nucleotides and calcium. CK-2 phosphorylates many different
proteins. The substrate specificity [1] of this enzyme can be
summarized as follows: (1) Under comparable conditions Ser is
favored over Thr.; (2) An acidic residue (either Asp or Glu) must
be present three residues from the C-terminal of the phosphate
acceptor site; (3) Additional acidic residues in positions +1, +2,
+4, and +5 increase the phosphorylation rate. Most physiological
substrates have at least one acidic residue in these positions; (4)
Asp is preferred to Glu as the provider of acidic determinants; and
(5) A basic residue at the N-terminal of the acceptor site
decreases the phosphorylation rate, while an acidic one will
increase it.
[0201] A consensus pattern for casein kinase II phosphorylations
site is as follows: [ST]-x(2)-[DE], wherein `x` represents any
amino acid, and S or T is the phosphorylation site.
[0202] Additional information specific to casein kinase II
phosphorylation site domains may be found in reference to the
following publication: Pinna L. A., Biochim. Biophys. Acta
1054:267-284(1990); which is hereby incorporated herein in its
entirety.
[0203] In preferred embodiments, the following casein kinase II
phosphorylation site polypeptide is encompassed by the present
invention: SLGNQSVVEPNIAI (SEQ ID NO:42), STFIHTNVDGLNPD (SEQ ID
NO:43), QKIISSKTDENEQD (SEQ ID NO:44), VYWNLSAKDWDTYG (SEQ ID
NO:45), KNLQTSDGDINNID (SEQ ID NO:46), LRSLPTLHERFRLL (SEQ ID
NO:47), LETSPSTEEITLSE (SEQ ID NO:48), STEEITLSESDNAK (SEQ ID
NO:49), and/or EEITLSESDNAKES (SEQ ID NO:50). Polynucleotides
encoding these polypeptides are also provided. The present
invention also encompasses the use of this casein kinase II
phosphorylation site polypeptide as an immunogenic and/or antigenic
epitope as described elsewhere herein.
[0204] The HGPRBMY6 polypeptide was predicted to comprise three
cAMP- and cGMP-dependent protein kinase phosphorylation site using
the Motif algorithm (Genetics Computer Group, Inc.). There has been
a number of studies relative to the specificity of cAMP- and
cGMP-dependent protein kinases. Both types of kinases appear to
share a preference for the phosphorylation of serine or threonine
residues found close to at least two consecutive N-terminal basic
residues.
[0205] A consensus pattern for cAMP- and cGMP-dependent protein
kinase phosphorylation sites is as follows: [RK](2)-x-[ST], wherein
"x" represents any amino acid, and S or T is the phosphorylation
site.
[0206] Additional information specific to cAMP- and cGMP-dependent
protein kinase phosphorylation sites may be found in reference to
the following publication: Fremisco J. R., Glass D. B., Krebs E. G,
J. Biol. Chem. 255:4240-4245(1980); Glass D. B., Smith S. B., J.
Biol. Chem. 258:14797-14803(1983); and Glass D. B., El-Maghrabi M.
R., Pilkis S. J., J. Biol. Chem. 261:2987-2993(1986); which is
hereby incorporated herein in its entirety.
[0207] In preferred embodiments, the following cAMP- and
cGMP-dependent protein kinase phosphorylation site polypeptides are
encompassed by the present invention: VTRKVRKTSVTWVL (SEQ ID
NO:51), NLTSTKKVSSMKKI (SEQ ID NO:52), and/or LSSIGRRKSLPSVT (SEQ
ID NO:53). Polynucleotides encoding this polypeptide are also
provided. The present invention also encompasses the use of these
cAMP- and cGMP-dependent protein kinase phosphorylation site
polypeptides as immunogenic and/or antigenic epitopes as described
elsewhere herein.
[0208] The HGPRBMY6 polypeptide has been shown to comprise eight
glycosylation sites according to the Motif algorithm (Genetics
Computer Group, Inc.). As discussed more specifically herein,
protein glycosylation is thought to serve a variety of functions
including: augmentation of protein folding, inhibition of protein
aggregation, regulation of intracellular trafficking to organelles,
increasing resistance to proteolysis, modulation of protein
antigenicity, and mediation of intercellular adhesion.
[0209] Asparagine glycosylation sites have the following concensus
pattern, N-{P}-[ST]-{P}, wherein N represents the glycosylation
site. However, it is well known that that potential N-glycosylation
sites are specific to the consensus sequence Asn-Xaa-Ser/Thr.
However, the presence of the consensus tripeptide is not sufficient
to conclude that an asparagine residue is glycosylated, due to the
fact that the folding of the protein plays an important role in the
regulation of N-glycosylation. It has been shown that the presence
of proline between Asn and Ser/Thr will inhibit N-glycosylation;
this has been confirmed by a recent statistical analysis of
glycosylation sites, which also shows that about 50% of the sites
that have a proline C-terminal to Ser/Thr are not glycosylated.
Additional information relating to asparagine glycosylation may be
found in reference to the following publications, which are hereby
incorporated by reference herein: Marshall R. D., Annu. Rev.
Biochem. 41:673-702(1972); Pless D. D., Lennarz W. J., Proc. Natl.
Acad. Sci. U.S.A. 74:134-138(1977); Bause E., Biochem. J.
209:331-336(1983); Gavel Y., von Heijne G., Protein Ens.
3:433-442(1990); and Miletich J. P., Broze G. J. Jr., J. Biol.
Chem. 265:11397-11404(1990).
[0210] In preferred embodiments, the following asparagine
glycosylation site polypeptides are encompassed by the present
invention: SLSLGNQSVVEPNI (SEQ ID NO:54), AIQSANFSSENAVG (SEQ ID
NO:55), LQVLLNMTKNYTKT (SEQ ID NO:56), LNMTKNYTKTCGFV (SEQ ID
NO:57), ACVYWNLSAKDWDT (SEQ ID NO:58), LRCRCNHTTNFAVL (SEQ ID
NO:59), WKNNQNLTSTKKVS (SEQ ID NO:60), and/or IFCLFNTTQGLQIF (SEQ
ID NO:61). Polynucleotides encoding these polypeptides are also
provided. The present invention also encompasses the use of these
HGPRBMY6 asparagine glycosylation site polypeptide as imnmunogenic
and/or antigenic epitopes as described elsewhere herein.
[0211] The HGPRBMY6 polypeptide was predicted to comprise five
N-myristoylation sites using the Motif algorithm (Genetics Computer
Group, Inc.). An appreciable number of eukaryotic proteins are
acylated by the covalent addition of myristate (a C14-saturated
fatty acid) to their N-terminal residue via an amide linkage. The
sequence specificity of the enzyme responsible for this
modification, myristoyl CoA:protein N-myristoyl transferase (NMT),
has been derived from the sequence of known N-myristoylated
proteins and from studies using synthetic peptides. The specificity
seems to be the following: i.) The N-terminal residue must be
glycine; ii.) In position 2, uncharged residues are allowed; iii.)
Charged residues, proline and large hydrophobic residues are not
allowed; iv.) In positions 3 and 4, most, if not all, residues are
allowed; v.) In position 5, small uncharged residues are allowed
(Ala, Ser, Thr, Cys, Asn and Gly). Serine is favored; and vi.) In
position 6, proline is not allowed.
[0212] A consensus pattern for N-myristoylation is as follows:
G-{EDRKHPFYW}-x(2)-[STAGCN]-{P}, wherein `x` represents any amino
acid, and G is the N-myristoylation site.
[0213] Additional information specific to N-myristoylation sites
may be found in reference to the following publication: Towler D.
A., Gordon J. I., Adams S. P., Glaser L., Annu. Rev. Biochem.
57:69-99(1988); and Grand R. J. A., Biochem. J. 258:625-638(1989);
which is hereby incorporated herein in its entirety.
[0214] In preferred embodiments, the following N-myristoylation
site polypeptides are encompassed by the present invention:
FSVQKGASSSLVSSST (SEQ ID NO:62), ILSNVGCALSVTGLAL (SEQ ID NO:63),
ALSVTGLALTVIFQIV (SEQ ID NO:64), LLFVFGIENSNKNLQT (SEQ ID NO:65),
and/or VAITVGVIYSQNGNNP (SEQ ID NO:66). Polynucleotides encoding
these polypeptides are also provided. The present invention also
encompasses the use of these N-myristoylation site polypeptides as
immunogenic and/or antigenic epitopes as described elsewhere
herein.
[0215] G-protein coupled receptors (also called R7G) are an
extensive group of hormones, neurotransmitters, odorants and light
receptors which transduce extracellular signals by interaction with
guanine nucleotide-binding (G) proteins. Some examples of receptors
that belong to this family are provided as follows:
5-hydroxytryptamine (serotonin) 1A to 1F, 2A to 2C, 4, 5A, 5B, 6
and 7, Acetylcholine, muscarinic-type, M1 to M5, Adenosine A1, A2A,
A2B and A3, Adrenergic alpha-1A to -1C; alpha-2A to -2D; beta-1 to
-3, Angiotensin II types I and II, Bombesin subtypes 3 and 4,
Bradykinin B1 and B2, c3a and C5a anaphylatoxin, Cannabinoid CB1
and CB2, Chemokines C-C CC-CKR-1 to CC-CKR-8, Chemokines C-X-C
CXC-CKR-1 to CXC-CKR-4, Cholecystokinin-A and
cholecystokinin-B/gastrin, Dopamine D1 to D5, Endothelin ET-a and
ET-b, fMet-Leu-Phe (fMLP) (N-fornyl peptide), Follicle stimulating
hormone (FSH-R), Galanin, Gastrin-releasing peptide (GRP-R),
Gonadotropin-releasing hormone (GNRH-R), Histamine H1 and H2
(gastric receptor I), Lutropin-choriogonadotropic hormone (LSH-R),
Melanocortin MC1R to MC5R, Melatonin, Neuromedin B (NMB-R),
Neuromedin K (NK-3R), Neuropeptide Y types 1 to 6, Neurotensin
(NT-R), Octopamine (tyramine) from insects, Odorants, Opioids
delta-, kappa- and mu-types, Oxytocin (OT-R), Platelet activating
factor (PAF-R), Prostacyclin, Prostaglandin D2, Prostaglandin E2,
EP1 to EP4 subtypes, Prostaglandin F2, Purinoreceptors (ATP),
Somatostatin types 1 to 5, Substance-K (NK-2R), Substance-P
(NK-1R), Thrombin, Thromboxane A2, Thyrotropin (TSH-R), Thyrotropin
releasing factor (TRH-R), Vasopressin V1a, V1b and V2, Visual
pigments (opsins and rhodopsin), Proto-oncogene mas, Caenorhabditis
elegans putative receptors C06G4.5, C38C10.1, C43C3.2, T27D1.3 and
ZC84.4, Three putative receptors encoded in the genome of
cytomegalovirus: US27, US28, and UL33., ECRF3, a putative receptor
encoded in the genome of herpesvirus saimiri.
[0216] The structure of all GPCRs are thought to be identical. They
have seven hydrophobic regions, each of which most probably spans
the membrane. The N-terminus is located on the extracellular side
of the membrane and is often glycosylated, while the C-terminus is
cytoplasmic and generally phosphorylated. Three extracellular loops
alternate with three intracellular loops to link the seven
transmembrane regions. Most, but not all of these receptors, lack a
signal peptide. The most conserved parts of these proteins are the
transmembrane regions and the first two cytoplasmic loops. A
conserved acidic-Arg-aromatic triplet is present in the N-terminal
extremity of the second cytoplasmic loop and could be implicated in
the interaction with G proteins.
[0217] The putative concensus sequence for GPCRs comprises the
conserved triplet and also spans the major part of the third
transmembrane helix, and is as follows:
[GSTALIVMFYWC]-[GSTANCPDE]-{EDPKRH}-x(2)-[LIVMNQGA]-x(-
2)-[LIVMFT]-[GSTANC]-[LIVMFYWSTAC]-[DENH]-R-[FYWCSH]-x(2)-[LIVM],
where "X" represents any amino acid.
[0218] Additional information relating to G-protein coupled
receptors may be found in reference to the following publications:
Strosberg A. D., Eur. J. Biochem. 196:1-10(1991); Kerlavage A. R.,
Curr. Opin. Struct. Biol. 1:394-401(1991); Probst W. C., Snyder L.
A., Schuster D. I., Brosius J., Sealfon S. C., DNA Cell Biol.
11:1-20(1992); Savarese T. M., Fraser C. M., Biochem. J.
283:1-9(1992); Branchek T., Curr. Biol. 3:315-317(1993); Stiles G.
L., J. Biol. Chem. 267:6451-6454(1992); Friell T., Kobilka B. K.,
Lefkowitz R. J., Caron M. G., Trends Neurosci. 11:321-324(1988);
Stevens C. F., Curr. Biol. 1:20-22(1991); Sakurai T., Yanagisawa
M., Masaki T., Trends Pharmacol. Sci. 13:103-107(1992); Salesse R.,
Remy J. J., Levin J. M., Jallal B., Gamier J., Biochimic
73:109-120(1991); Lancet D., Ben-Arie N., Curr. Biol.
3:668-674(1993); Uhl G. R., Childers S., Pasternak G., Trends
Neurosci. 17:89-93(1994); Barnard E. A., Burmstock G., Webb T. E.,
Trends Pharmacol. Sci. 15:67-70(1994); Applebury M. L., Hargrave P.
A., Vision Res. 26:1881-1895(1986); Attwood T. K., Eliopoulos E.
E., Findlay J. B. C., Gene 98:153-159(1991);
http://www.gcrdb.uthscsa.edu/; and
http://swift.embl-heidelberg.de/7tm/.
[0219] For the production of antibodies, various hosts including
goats, rabbits, sheep, rats, mice, humans, and others, can be
immunized by injection with HGPRBMY6 polypeptide, or any fragment
or oligopeptide thereof, which has immunogenic properties.
Depending on the host species, various adjuvants may be used to
increase the immunological response. Non-limiting examples of
suitable adjuvants include Freund's (incomplete), mineral gels such
as aluminum hydroxide or silica, and surface active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, KLH, and dinitrophenol. Adjuvants typically used in
humans include BCG (bacilli Calmette Gurin) and Corynebacterium
parvumn.
[0220] Preferably, the peptides, fragments, or oligopeptides used
to induce antibodies to HGPRBMY6 polypeptide (i.e., immunogens)
have an amino acid sequence having at least five amino acids, and
more preferably, at least 7-10 amino acids. It is also preferable
that the immunogens are identical to a portion of the amino acid
sequence of the natural protein; they may also contain the entire
amino acid sequence of a small, naturally occurring molecule. The
peptides, fragments or oligopeptides may comprise a single epitope
or antigenic determinant or multiple epitopes. Short stretches of
HGPRBMY6 amino acids may be fused with those of another protein,
such as KLH, and antibodies are produced against the chimeric
molecule.
[0221] Monoclonal antibodies to HGPRBMY6 polypeptide, or
immunogenic fragments thereof, may be prepared using any technique
which provides for the production of antibody molecules by
continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique (G. Kohler et al., 1975,
Nature, 256:495-497; D. Kozbor et al., 1985, J. Immunol. Methods,
81:31-42; R. J. Cote et al., 1983, Proc. Natl. Acad. Sci. USA,
80:2026-2030; and S. P. Cole et al., 1984, Mol. Cell Biol.,
62:109-120). The production of monoclonal antibodies is well known
and routinely used in the art.
[0222] In addition, techniques developed for the production of
"chimeric antibodies," the splicing of mouse antibody genes to
human antibody genes to obtain a molecule with appropriate antigen
specificity and biological activity can be used (S. L. Morrison et
al., 1984, Proc. Natl. Acad. Sci. USA, 81:6851-6855; M. S.
Neuberger et al., 1984, Nature, 312:604-608; and S. Takeda et al.,
1985, Nature, 314:452-454). Alternatively, techniques described for
the production of single chain antibodies may be adapted, using
methods known in the art, to produce HGPRBMY6 polypeptide-specific
single chain antibodies. Antibodies with related specificity, but
of distinct idiotypic composition, may be generated by chain
shuffling from random combinatorial immunoglobulin libraries (D. R.
Burton, 1991, Proc. Natl. Acad. Sci. USA, 88:11120-3). Antibodies
may also be produced by inducing in vivo production in the
lymphocyte population or by screening recombinant immunoglobulin
libraries or panels of highly specific binding reagents as
disclosed in the literature (R. Orlandi et al., 1989, Proc. Natl.
Acad. Sci. USA, 86:3833-3837 and G. Winter et al., 1991, Nature,
349:293-299).
[0223] Antibody fragments, which contain specific binding sites for
HGPRBMY6 polypeptide, may also be generated. For example, such
fragments include, but are not limited to, F(ab').sub.2 fragments
which can be produced by pepsin digestion of the antibody molecule
and Fab fragments which can be generated by reducing the disulfide
bridges of the F(ab').sub.2 fragments. Alternatively, Fab
expression libraries may be constructed to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity (W. D. Huse et al., 1989, Science, 254.1275-1281).
[0224] Various immunoassays can be used for screening to identify
antibodies having the desired specificity. Numerous protocols for
competitive binding or immunoradiometric assays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art. Such immunoassays typically involve
measuring the formation of complexes between HGPRBMY6 polypeptide
and its specific antibody. A two-site, monoclonal-based immunoassay
utilizing monoclonal antibodies reactive with two non-interfering
HGPRBMY6 polypeptide epitopes is preferred, but a competitive
binding assay may also be employed (Maddox, supra).
[0225] Another aspect of the invention relates to a method for
inducing an immunological response in a mammal which comprises
inoculating the mammal with HGPRBMY6 polypeptide, or a fragment
thereof, adequate to produce antibody and/or T cell immune response
to protect said animal from infections such as bacterial, fimgal,
protozoan and viral infections, particularly infections caused by
HIV-1 or HIV-2. Yet another aspect of the invention relates to a
method of inducing immunological response in a mammal which
comprises, delivering HGPRBMY6 polypeptide via a vector directing
expression of HGPRBMY6 polynucleotide in vivo in order to induce
such an immunological response to produce antibody to protect said
animal from diseases.
[0226] A further aspect of the invention relates to an
immunological/vaccine formulation (composition) which, when
introduced into a mammalian host, induces an immunological response
in that mammal to an HGPRBMY6 polypeptide wherein the composition
comprises an HGPRBMY6 polypeptide or HGPRBMY6 gene. The vaccine
formulation may fuirther comprise a suitable carrier. Since the
HGPRBMY6 polypeptide may be broken down in the stomach, it is
preferably administered parenterally (including subcutaneous,
intramuscular, intravenous, intradermal, etc., injection).
Formulations suitable for parenteral administration include aqueous
and non-aqueous sterile injection solutions which may contain
anti-oxidants, buffers, bacteriostats and solutes which render the
formulation isotonic with the blood of the recipient; and aqueous
and non-aqueous sterile suspensions which may include suspending
agents or thickening agents. The formulations may be presented in
unit-dose or multi-dose containers, for example, sealed ampoules
and vials, and may be stored in a freeze-dried condition requiring
only the addition of the sterile liquid carrier immediately prior
to use. The vaccine formulation may also include adjuvant systems
for enhancing the immunogenicity of the formulation, such as
oil-in-water systems and other systems known in the art. The dosage
will depend on the specific activity of the vaccine and can be
readily determined by routine experimentation.
[0227] In an embodiment of the present invention, the
polynucleotide encoding the HGPRBMY6 polypeptide, or any fragment
or complement thereof, may be used for therapeutic purposes. In one
aspect, antisense, to the polynucleotide encoding the HGPRBMY6
polypeptide, may be used in situations in which it would be
desirable to block the transcription of the mRNA. In particular,
cells may be transformed with sequences complementary to
polynucleotides encoding HGPRBMY6 polypeptide. Thus, complementary
molecules may be used to modulate HGPRBMY6 polynucleotide and
polypeptide activity, or to achieve regulation of gene function.
Such technology is now well known in the art, and sense or
antisense oligomers or oligonucleotides, or larger fragments, can
be designed from various locations along the coding or control
regions of polynucleotide sequences encoding HGPRBMY6
polypeptide.
[0228] Expression vectors derived from retroviruses, adenovirus,
herpes or vaccinia viruses, or from various bacterial plasmids may
be used for delivery of anucleotide sequences to the targeted
organ, tissue or cell population. Methods, which are well known to
those skilled in the art, can be used to construct recombinant
vectors which will express a nucleic acid sequence that is
complementary to the nucleic acid sequence encoding the HGPRBMY6
polypeptide. These techniques are described both in J. Sambrook et
al., supra and in F. M. Ausubel et al., supra.
[0229] Polypeptides used in treatment can also be generated
endogenously in the subject, in treatment modalities often referred
to a "gene therapy". Thus, for example, cells from a subject may be
engineered with a polynucleotide, such as DNA or RNA, to encode a
polypeptide ex vivo, and for example, by the use of a retroviral
plasmid vector. The cells can then be introduced into the
subject.
[0230] Transforming a cell or tissue with an expression vector that
expresses high levels of an HGPRBMY6 polypeptide-encoding
polynucleotide, or a fragment thereof can turn off the genes
encoding the HGPRBMY6 polypeptide. Such constructs may be used to
introduce untranslatable sense or antisense sequences into a cell.
Even in the absence of integration into the DNA, such vectors may
continue to transcribe RNA molecules until they are disabled by
endogenous nucleases. Transient expression may last for a month or
more with a non-replicating vector, and even longer if appropriate
replication elements are designed to be part of the vector
system.
[0231] Designing antisense molecules or complementary nucleic acid
can obtain modifications of gene expression sequences (DNA, RNA, or
PNA), to the control, 5', or regulatory regions of the gene
encoding the HGPRBMY6 polypeptide, (e.g., signal sequence,
promoters, enhancers, and introns). Oligonucleotides derived from
the transcription initiation site, e.g., between positions -10 and
+10 from the start site, are preferred. Similarly, inhibition can
be achieved using "triple helix" base-pairing methodology. Triple
helix pairing is useful because it causes inhibition of the ability
of the double helix to open sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using triplex DNA have been described (see,
for example, J. E. Gee et al., 1994, In: B. E. Huber and B. I.
Carr, Molecular and Immunologic Approaches, Futura Publishing Co.,
Mt. Kisco, N.Y.). The antisense molecule or complementary sequence
may also be designed to block translation of mRNA by preventing the
transcript from binding to ribosomes.
[0232] Ribozymes, i.e., enzymatic RNA molecules, may also be used
to catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. Suitable examples include engineered hammerhead motif
ribozyme molecules that can specifically and efficiently catalyze
endonucleolytic cleavage of sequences encoding HGPRBMY6
polypeptide.
[0233] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites which include the following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15
and 20 ribonucleotides corresponding to the region of the target
gene containing the cleavage site may be evaluated for secondary
structural features which may render the oligonucleotide
inoperable. The suitability of candidate targets may also be
evaluated by testing accessibility to hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0234] Complementary ribonucleic acid molecules and ribozymes
according to the invention may be prepared by any method known in
the art for the synthesis of nucleic acid molecules. Such methods
include techniques for chemically synthesizing oligonucleotides,
for example, solid phase phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in
vivo transcription of DNA sequences encoding HGPRBMY6. Such DNA
sequences may be incorporated into a wide variety of vectors with
suitable RNA polymerase promoters such as T7 or SP. Alternatively,
the cDNA constructs that constitutively or inducibly synthesize
complementary RNA can be introduced into cell lines, cells, or
tissues.
[0235] RNA molecules may be modified to increase intracellular
stability and half-life. Possible modifications include, but are
not limited to, the addition of flanking sequences at the 5' and/or
3' ends of the molecule, or the use of phosphorothioate or 2'
O-methyl, rather than phosphodiesterase linkages within the
backbone of the molecule. This concept is inherent in the
production of PNAs and can be extended in all of these molecules by
the inclusion of nontraditional bases such as inosine, queosine,
and wybutosine, as well as acetyl-, methyl-, thio-, and similarly
modified forms of adenine, cytosine, guanine, thymine, and uridine
which are not as easily recognized by endogenous endonucleases.
[0236] Many methods for introducing vectors into cells or tissues
are available and are equally suitable for use in vivo, in vitro,
and ex vivo. For ex vivo therapy, vectors may be introduced into
stem cells taken from the patient and clonally propagated for
autologous transplant back into that same patient. Delivery by
transfection and by liposome injections may be achieved using
methods, which are well known in the art.
[0237] Any of the therapeutic methods described above may be
applied to any individual in need of such therapy, including, for
example, mammals such as dogs, cats, cows, horses, rabbits,
monkeys, and most preferably, humans.
[0238] A further embodiment of the present invention embraces the
administration of a pharmaceutical composition, in conjunction with
a pharmaceutically acceptable carrier, diluent, or excipient, for
any of the above-described therapeutic uses and effects. Such
pharmaceutical compositions may comprise HGPRBMY6 nucleic acid,
polypeptide, or peptides, antibodies to HGPRBMY6 polypeptide,
mimetics, agonists, antagonists, or inhibitors of IHGPRBMY6
polypeptide or polynucleotide. The compositions may be administered
alone or in combination with at least one other agent, such as a
stabilizing compound, which may be administered in any sterile,
biocompatible pharmaceutical carrier, including, but not limited
to, saline, buffered saline, dextrose, and water. The compositions
may be administered to a patient alone, or in combination with
other agents, drugs, hormones, or biological response
modifiers.
[0239] The pharmaceutical compositions for use in the present
invention can be administered by any number of routes including,
but not limited to, oral, intravenous, intramuscular,
intra-arterial, intramedullary, intrathecal, intraventricular,
transdermal, subcutaneous, intraperitoneal, intranasal, enteral,
topical, sublingual, vaginal, or rectal means.
[0240] In addition to the active ingredients (i.e., the HGPRBMY6
nucleic acid or polypeptide, or functional fragments thereof), the
pharmaceutical compositions may contain suitable pharmaceutically
acceptable carriers or excipients comprising auxiliaries which
facilitate processing of the active compounds into preparations
which can be used pharmaceutically. Further details on techniques
for formulation and administration are provided in the latest
edition of Remington's Pharmaceutical Sciences (Maack Publishing
Co., Easton, Pa.).
[0241] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the patient.
[0242] Pharmaceutical preparations for oral use can be obtained by
the combination of active compounds with solid excipient,
optionally grinding a resulting mixture, and processing the mixture
of granules, after adding suitable auxiliaries, if desired, to
obtain tablets or dragee cores. Suitable excipients are
carbohydrate or protein fillers, such as sugars, including lactose,
sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,
potato, or other plants; cellulose, such as methyl cellulose,
hydroxypropyl-methylcellulose, or sodium carboxymethylcellulose;
gums, including arabic and tragacanth, and proteins such as gelatin
and collagen. If desired, disintegrating or solubilizing agents may
be added, such as cross-linked polyvinyl pyrrolidone, agar, alginic
acid, or a physiologically acceptable salt thereof, such as sodium
alginate.
[0243] Dragee cores may be used in conjunction with physiologically
suitable coatings, such as concentrated sugar solutions, which may
also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for product
identification, or to characterize the quantity of active compound,
i.e., dosage.
[0244] Pharmaceutical preparations, which can be used orally,
include push-fit capsules made of gelatin, as well as soft, scaled
capsules made of gelatin and a coating, such as glycerol or
sorbitol. Push-fit capsules can contain active ingredients mixed
with a filler or binders, such as lactose or starches, lubricants,
such as talc or magnesium stearate, and, optionally, stabilizers.
In soft capsules, the active compounds may be dissolved or
suspended in suitable liquids, such as fatty oils, liquid, or
liquid polyethylene glycol with or without stabilizers.
[0245] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances, which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. In addition, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acid esters, such as
ethyloleate or triglycerides, or liposomes. Optionally, the
suspension may also contain suitable stabilizers or agents who
increase the solubility of the compounds to allow for the
preparation of highly concentrated solutions.
[0246] For topical or nasal administration, penetrants or
permeation agents that are appropriate to the particular barrier to
be permeated are used in the formulation. Such penetrants are
generally known in the art.
[0247] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes.
[0248] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic,
and the like. Salts tend to be more soluble in aqueous solvents, or
other protonic solvents, than are the corresponding free base
forms. In other cases, the preferred preparation may be a
lyophilized powder which may contain any or all of the following:
1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH
range of 4.5 to 5.5, combined with a buffer prior to use. After the
pharmaceutical compositions have been prepared, they can be placed
in an appropriate container and labeled for treatment of an
indicated condition. For administration of HGPRBMY6 product, such
labeling would include amount, frequency, and method of
administration.
[0249] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
The determination of an effective dose or amount is well within the
capability of those skilled in the art. For any compound, the
therapeutically effective dose can be estimated initially either in
cell culture assays, e.g., using neoplastic cells, or in animal
models, usually mice, rabbits, dogs, or pigs. The animal model may
also be used to determine the appropriate concentration range and
route of administration. Such information can then be used and
extrapolated to determine useful doses and routes for
administration in humans.
[0250] A therapeutically effective dose refers to that amount of
active ingredient, for example, HGPRBMY6 polypeptide, or fragments
thereof, antibodies to HGPRBMY6 polypeptide, agonists, antagonists
or inhibitors of HGPRBMY6 polypeptide, which ameliorates, reduces,
or eliminates the symptoms or condition. Therapeutic efficacy and
toxicity may be determined by standard pharmaceutical procedures in
cell cultures or experimental animals, e.g., ED.sub.50 (the dose
therapeutically effective in 50% of the population) and LD.sub.50
(the dose lethal to 50% of the population). The dose ratio of toxic
to therapeutic effects is the therapeutic index, which can be
expressed as the ratio, ED.sub.50/LD.sub.50. Pharmaceutical
compositions, which exhibit large therapeutic indices, are
preferred. The data obtained from cell culture assays and animal
studies are used in determining a range of dosages for human use.
Preferred dosage contained in a pharmaceutical composition is
within a range of circulating concentrations that include the
ED.sub.50 with little or no toxicity. The dosage varies within this
range depending upon the dosage form employed, sensitivity of the
patient, and the route of administration.
[0251] The practitioner, who will consider the factors related to
the individual requiring treatment, will determine the exact
dosage. Dosage and administration are adjusted to provide
sufficient levels of the active moiety or to maintain the desired
effect. Factors, which may be taken into account, include the
severity of the individual's disease state, general health of the
patient, age, weight, and gender of the patient, diet, time and
frequency of administration, drug combination(s), reaction
sensitivities, and tolerance/response to therapy. As a general
guide, long-acting pharmaceutical compositions may be administered
every 3 to 4 days, every week, or once every two weeks, depending
on half-life and clearance rate of the particular formulation.
Variations in the dosage levels can be adjusted using standard
empirical routines for optimization, as is well understood in the
art.
[0252] Normal dosage amounts may vary from 0.1 to 100,000
micrograms (.mu.g), up to a total dose of about 1 gram (g),
depending upon the route of administration. Guidance as to
particular dosages and methods of delivery is provided in the
literature and is generally available to practitioners in the art.
Those skilled in the art will employ different formulations for
nucleotides than for proteins or their inhibitors. Similarly,
delivery of polynucleotides or polypeptides will be specific to
particular cells, conditions, locations, and the like.
[0253] In another embodiment of the present invention, antibodies
which specifically bind to the HGPRBMY6 polypeptide may be used for
the diagnosis of conditions or diseases characterized by expression
(or overexpression) of the HGPRBMY6 polynucleotide or polypeptide,
or in assays to monitor patients being treated with the HGPRBMY6
polypeptide, or its agonists, antagonists, or inhibitors. The
antibodies useful for diagnostic purposes may be prepared in the
same manner as those described above for use in therapeutic
methods. Diagnostic assays for the HGPRBMY6 polypeptide include
methods, which utilize the antibody and a label to detect the
protein in human body fluids or extracts of cells or tissues. The
antibodies may be used with or without modification, and may be
labeled by joining them, either covalently or non-covalently, with
a reporter molecule. A wide variety of reporter molecules, which
are known in the art may be used, several of which are described
above.
[0254] The use of mammalian cell reporter assays to demonstrate
functional coupling of known GPCRs (G Protein Coupled Receptors)
has been well documented in the literature (Gilman, 1987, Boss et
al., 1996; Alam & Cook, 1990; George et al., 1997; Selbie &
Hill, 1998; Rees et al., 1999). In fact, reporter assays have been
successfully used for identifying novel small molecule agonists or
antagonists against GPCRs as a class of drug targets (Zlokamik et
al., 1998; George et al., 1997; Boss et al., 1996; Rees et al,
2001). In such reporter assays, a promoter is regulated as a direct
consequence of activation of specific signal transduction cascades
following agonist binding to a GPCR (Alam & Cook 1990; Selbie
& Hill, 1998; Boss et al., 1996; George et al., 1997; Gilman,
1987).
[0255] A number of response element-based reporter systems have
been developed that enable the study of GPCR function. These
include cAMP response element (CRE)-based reporter genes for G
alpha i/o, G alpha s-coupled GPCRs, Nuclear Factor Activator of
Transcription (NFAT)-based reporters for G alpha q/11 or the
promiscuous G protein G alpha 15/16-coupled receptors and MAP
kinase reporter genes for use in G alpha i/o coupled receptors
(Selbie & Hill, 1998; Boss et al., 1996; George et al., 1997;
Blahos, et al., 2001; Offermann & Simon, 1995; Gilman, 1987;
Rees et al., 2001). Transcriptional response elements that regulate
the expression of Beta-Lactamase within a CHO K1 cell line
(CHO-NFAT/CRE: Aurora Biosciences.TM.) (Zlokamik et al., 1998) have
been implemented to characterize the function of the orphan
HGPRBMY6 polypeptide of the present invention. The system enables
demonstration of constitutive G-protein coupling to endogenous
cellular signaling components upon intracellular overexpression of
orphan receptors. Overexpression has been shown to represent a
physiologically relevant event. For example, it has been shown that
overexpression occurs in nature during metastatic carcinomas,
wherein defective expression of the monocyte chemotactic protein 1
receptor, CCF2, in macrophages is associated with the incidence of
human ovarian carcinoma (Sica, et al., 2000; Salcedo et al., 2000).
Indeed, it has been shown that overproduction of the Beta 2
Adrenergic Receptor in transgenic mice leads to constitutive
activation of the receptor signaling pathway such that these mice
exhibit increased cardiac output (Kypson et al., 1999; Dorn et al.,
1999). These are only a few of the many examples demonstrating
constitutive activation of GPCRs whereby many of these receptors
are likely to be in the active, R*, conformation (J. Wess 1997)
(Example 6).
[0256] Several assay protocols including ELISA, RIA, and FACS for
measuring HGPRBMY6 polypeptide are known in the art and provide a
basis for diagnosing altered or abnormal levels of HGPRBMY6
polypeptide expression. Normal or standard values for HGPRBMY6
polypeptide expression are established by combining body fluids or
cell extracts taken from normal mammalian subjects, preferably
human, with antibody to the HGPRBMY6 polypeptide under conditions
suitable for complex formation. The amount of standard complex
formation may be quantified by various methods; photometric means
are preferred. Quantities of HGPRBMY6 polypeptide expressed in
subject sample, control sample, and disease samples from biopsied
tissues are compared with the standard values. Deviation between
standard and subject values establishes the parameters for
diagnosing disease.
[0257] Microarravs and Screening Assays
[0258] In another embodiment of the present invention,
oligonucleotides, or longer fragments derived from the HGPRBMY6
polynucleotide sequence described herein may be used as targets in
a microarray. The microarray can be used to monitor the expression
level of large numbers of genes simultaneously (to produce a
transcript image), and to identify genetic variants, mutations and
polymorphisms. This information may be used to determine gene
function, to understand the genetic basis of a disease, to diagnose
disease, and to develop and monitor the activities of therapeutic
agents. In a particular aspect, the microarray is prepared and used
according to the methods described in WO 95/11995 (Chee et al.); D.
J. Lockhart et al., 1996, Nature Biotechnology, 14:1675-1680; and
M. Schena et al., 1996, Proc. Natl. Acad. Sci. USA,
93:10614-10619). Microarrays are further described in U.S. Pat. No.
6,015,702 to P. Lal et al.
[0259] In another embodiment of this invention, the nucleic acid
sequence, which encodes the HGPRBMY6 polypeptide may also be used
to generate hybridization probes, which are useful for mapping the
naturally occurring genomic sequence. The sequences may be mapped
to a particular chromosome, to a specific region of a chromosome,
or to artificial chromosome constructions (HACs), yeast artificial
chromosomes (YACs), bacterial artificial chromosomes (BACs),
bacterial PI constructions, or single chromosome cDNA libraries, as
reviewed by C. M. Price, 1993, Blood Rev., 7:127-134 and by B. J.
Trask, 1991, Trends Genet., 7:149-154.
[0260] Fluorescent In Situ Hybridization (FISH), (as described in
I. Verma et al., 1988, Human Chromosomes: A Manual of Basic
Techniques Pergamon Press, New York, N.Y.) may be correlated with
other physical chromosome mapping techniques and genetic map data.
Examples of genetic map data can be found in numerous scientific
journals or at Online Mendelian Inheritance in Man (OMIM).
Correlation between the location of the gene encoding the HGPRBMY6
polypeptide on a physical chromosomal map and a specific disease,
or predisposition to a specific disease, may help delimit the
region of DNA associated with that genetic disease. The nucleotide
sequences, particularly that of SEQ ID NO:2, or fragments thereof,
according to this invention may be used to detect differences in
gene sequences between normal, carrier, or affected
individuals.
[0261] In situ hybridization of chromosomal preparations and
physical mapping techniques such as linkage analysis using
established chromosomal markers may be used for extending genetic
maps. Often the placement of a gene on the chromosome of another
mammalian species, such as mouse, may reveal associated markers,
even if the number or arm of a particular human chromosome is not
known. New sequences can be assigned to chromosomal arms, or parts
thereof, by physical mapping. This provides valuable information to
investigators searching for disease genes using positional cloning
or other gene discovery techniques. Once the disease or syndrome
has been crudely localized by genetic linkage to a particular
genomic region, for example, AT to 11q22-23 (R. A. Gatti et al.,
1988, Nature, 336:577-580), any sequences mapping to that area may
represent associated or regulatory genes for further investigation.
The nucleotide sequence of the present invention may also be used
to detect differences in the chromosomal location due to
translocation, inversion, and the like, among normal, carrier, or
affected individuals.
[0262] In another embodiment of the present invention, the HGPRBMY6
polypeptide, its catalytic or immunogenic fragments or
oligopeptides thereof, can be used for screening libraries of
compounds in any of a variety of drug screening techniques. The
fragment employed in such screening may be free in solution,
affixed to a solid support, borne on a cell surface, or located
intracellularly. The formation of binding complexes, between the
HGPRBMY6 polypeptide, or portion thereof, and the agent being
tested, may be measured utilizing techniques commonly practiced in
the art.
[0263] Another technique for drug screening which may be used
provides for high throughput screening of compounds having suitable
binding affinity to the protein of interest as described in WO
84/03564 (Venton, et al.). In this method, as applied to the
HGPRBMY6 protein, large numbers of different small test compounds
are synthesized on a solid substrate, such as plastic pins or some
other surface. The test compounds are reacted with the HGPRBMY6
polypeptide, or fragments thereof, and washed. Bound HGPRBMY6
polypeptide is then detected by methods well known in the art.
Purified HGPRBMY6 polypeptide can also be coated directly onto
plates for use in the aforementioned drug screening techniques.
Alternatively, non-neutralizing antibodies can be used to capture
the peptide and immobilize it on a solid support.
[0264] In a further embodiment of this invention, competitive drug
screening assays can be used in which neutralizing antibodies,
capable of binding the HGPRBMY6 polypeptide, specifically compete
with a test compound for binding to the HGPRBMY6 polypeptide. In
this manner, the antibodies can be used to detect the presence of
any peptide, which shares one or more antigenic determinants with
the HGPRBMY6 polypeptide.
EXAMPLES
[0265] The Examples herein are meant to exemplify the various
aspects of carrying out the invention and are not intended to limit
the scope of the invention in any way. The Examples do not include
detailed descriptions for conventional methods employed, such as in
the construction of vectors, the insertion of cDNA into such
vectors, or the introduction of the resulting vectors into the
appropriate host. Such methods are well known to those skilled in
the art and are described in numerous publication's, for example,
Sambrook, Fritsch, and Maniatis, Molecular Cloning: a Laboratory
Manual, 2.sup.nd Edition, Cold Spring Harbor Laboratory Press, USA,
(1989).
Example 1
Bioinformatics Analysis
[0266] G-protein coupled receptor sequences were used as a probe to
search the Incyte and public domain EST databases. The search
program used was gapped BLAST (S. F. Altschul, et al., Nuc. Acids
Res., 25:3389-4302 (1997)). The top EST hits from the BLAST results
were searched back against the non-redundant protein and patent
sequence databases. From this analysis, ESTs encoding potential
novel GPCRs were identified based on sequence homology. The Incyte
EST (CloneID: 2206642) was selected as a potential novel GPCR
candidate, called HGPRBMY6, for subsequent analysis. This EST was
sequenced and the full-length clone of this GPCR was obtained using
the EST sequence information and conventional methods. The complete
protein sequence of HGPRBMY6 was analyzed for potential
transmembrane domains. The TMPRED program (K. Hofmann and W.
Stoffel, Biol. Chem., 347:166 (1993)) was used for transmembrane
prediction. The predicted transmembrane (TM) domains of the
HGPRBMY6 match with similar predicted domains of related GPCRs at
the sequence level. Based on sequence, structure and known GPCR
signature sequences, the orphan protein, HGPRBMY6, is a novel human
GPCR.
Example 2
Cloning of the Novel Human GPCR HGPRBMY6
[0267] Using the EST sequence, an antisense 80 base pair
oligonucleotide with biotin on the 5' end was designed to be
complementary to the putative coding region of HGPRBMY6 as follows:
5'-b-GCT GTG CAG CGC TGA GTG CGT TCC AGG TAA ATG TCA CTA ACA GAA
AAT AGT GCA GTA AGG CGG CAA TCG CAG TGC ACA TG-3' (SEQ ID NO:5).
This biotinylated oligo was incubated with a mixture of
single-stranded covalently closed circular cDNA libraries which
contained DNA corresponding to the sense strand. Hybrids between
the biotinylated oligo and the circular cDNA were captured on
streptavidin magnetic beads. Upon thermal release of the cDNA from
the biotinylated oligo, the single stranded cDNA was converted into
double strands using a primer homologous to a sequence on the cDNA
cloning vector. The double stranded cDNA was introduced into E.
coli by electroporation and the resulting colonies were screened by
PCR, using a primer pair designed from the EST sequence to identify
the proper cDNA.
[0268] Oligos used to identify the cDNA by PCR were as follows:
HGPRBMY6s (SEQ ID NO:6) 5'-CAGACACCAT TAACATCCCG AAT-3'; and
HGPRBMY6a (SEQ ID NO:7) 5'-AGAATGAAAT GCCGAGGAAG AG-3'
[0269] Those cDNA clones that were positive by PCR had the inserts
sized and two of the largest clones (4.2 Kb and 3.5 Kb) were chosen
for DNA sequencing. Both clones had identical sequence over the
regions in common.
Example 3
Expression Profiling of Novel Human GPCR, HGPRBMY6
[0270] The same PCR primer pair used to identify HGPRBMY6 cDNA
clones (HGPRBMY6s- SEQ ID NO:6 and HGPRBMY6a- SEQ ID NO:7) was used
to measure the steady state levels of mRNA by quantitative PCR.
Briefly, first strand cDNA was made from commercially available
mRNA. The relative amount of cDNA used in each assay was determined
by performing a parallel experiment using a primer pair for the
cyclophilin gene, which is expressed in equal amounts in all
tissues. The cyclophilin primer pair detected small variations in
the amount of cDNA in each sample, and these data were used for
normalization of the data obtained with the primer pair for
HGPRBMY6. The PCR data were converted into a relative assessment of
the difference in transcript abundance among the tissues tested and
the data are presented in FIG. 7. Transcripts corresponding to the
orphan GPCR, HGPRBMY6, were found to be highly expressed in small
intestine.
Example 4
G-protein Coupled Receptor PCR Expression Profiling
[0271] RNA quantification was performed using the Taqman
real-time-PCR fluorogenic assay. The Taqman assay is one of the
most precise methods for assaying the concentration of nucleic acid
templates.
[0272] All cell lines were grown using standard conditions: RPMI
1640 supplemented with 10% fetal bovine serum, 100 IU/ml
penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine, 10 mM
Hepes (all from GibcoBRL; Rockville, Md.). Eighty percent confluent
cells were washed twice with phosphate-buffered saline (GibcoBRL)
and harvested using 0.25% trypsin (GibcoBRL). RNA was prepared
using the RNeasy Maxi Kit from Qiagen (Valencia, Calif.).
[0273] cDNA template for real-time PCR was generated using the
Superscript First Strand Synthesis system for RT-PCR.
[0274] SYBR Green real-time PCR reactions were prepared as follows:
The reaction mix consisted of 20 ng first strand cDNA; 50 nM
Forward Primer; 50 nM Reverse Primer; 0.75.times.SYBR Green I
(Sigma); 1.times.SYBR Green PCR Buffer (50 mM Tris-HCl pH8.3, 75 mM
KCl); 10% DMSO; 3 mM MgCl.sub.2; 300 M each dATP, dGTP, dTTP, dCTP;
1 U Platinum Taq DNA Polymerase High Fidelity (Cat #11304-029; Life
Technologies; Rockville, Md.); 1:50 dilution; ROX (Life
Technologies). Real-time PCR was performed using an Applied
Biosystems 5700 Sequence Detection System. Conditions were 95 C.
for 10 min (denaturation and activation of Platinum Taq DNA
Polymerase), 40 cycles of PCR (95 C. for 15 sec, 60 C. for 1 min).
PCR products are analyzed for uniform melting using an analysis
algorithm built into the 5700 Sequence Detection System.
[0275] Forward primer: 383: 5'-CAGACACCATTAACATCCCGAAT-3' (SEQ ID
NO:22); and
[0276] Reverse primer: 384: 5'-AGAATGAAATGCCGAGGAAGAG-3' (SEQ ID
NO:23).
[0277] cDNA quantification used in the normalization of template
quantity was performed using Taqman technology. Taqman reactions
are prepared as follows: The reaction mix consisted of 20 ng first
strand cDNA; 25 nM GAPDH-F3, Forward Primer; 250 nM GAPDH-R1
Reverse Primer; 200 nM GAPDH-PVIC Taqman Probe (fluorescent dye
labeled oligonucleotide primer); 1.times.Buffer A (Applied
Biosystems); 5.5 mM MgCl2; 300 M DATP, dGTP, dTTP, dCTP; 1 U
Amplitaq Gold (Applied Biosystems). GAPDH, D-glyceraldehyde
-3-phosphate dehydrogenase, was used as control to normalize mRNA
levels.
[0278] Real-time PCR was performed using an Applied Biosystems 7700
Sequence Detection System. Conditions were 95 C. for 10 min.
(denaturation and activation of Amplitaq Gold), 40 cycles of PCR
(95 C. for 15 sec, 60 C. for 1 min).
[0279] The sequences for the GAPDH oligonucleotides used in the
Taqman reactions are as follows:
[0280] GAPDH-F3-5'-AGCCGAGCCACATCGCT-3' (SEQ ID NO:24)
[0281] GAPDH-R1-5'-GTGACCAGGCGCCCAATAC-3' (SEQ ID NO:25)
[0282] GAPDH-PVIC Taqman
Probe-VIC-5'-CAAATCCGTTGACTCCGACCTTCACCTT-3' TAMRA (SEQ ID
NO:26).
[0283] The Sequence Detection System generates a Ct (threshold
cycle) value that is used to calculate a concentration for each
input cDNA template. cDNA levels for each gene of interest are
normalized to GAPDH cDNA levels to compensate for variations in
total cDNA quantity in the input sample. This is done by generating
GAPDH Ct values for each cell line. Ct values for the gene of
interest and GAPDH are inserted into a modified version of the Ct
equation (Applied Biosystems Prism 7700 Sequence Detection System
User Bulletin #2), which is used to calculate a GAPDH normalized
relative cDNA level for each specific cDNA. The Ct equation is as
follows: relative quantity of nucleic acid
template=2.sup.Ct=2.sup.(Cta-Ctb), where Cta=Ct target-Ct GAPDH,
and Ctb=Ct reference-Ct GAPDH. (No reference cell line was used for
the calculation of relative quantity; Ctb was defined as 21).
[0284] The Graph # of Table 1 corresponds to the tissue type
position number of FIG. 8. Interestingly, HGPRBMY6 (also known as
GPCR29) messenger RNA was found to be preferentially expressed in
colon tumor cell lines. The average colon cell line expresses BMY6
60-fold higher than the average BMY6 expression in non-colon tumor
cell lines assayed. Additionally, two of the colon tumor cell lines
express BMY6 600 to 800 (579-855)-fold greater than the average
expression in non-colon tumor cell lines in the OCLP-1 (oncology
cell line panel) assayed.
1TABLE 1 Graph Ct Ct # Name Tissue GAPDH GPCR29 dCt ddCt Quant. 1
AIN 4 breast 17.49 37.38 19.89 -1.11 2.2E+00 2 AIN 4T breast 17.15
38.2 21.05 0.05 9.7E-01 3 AIN4/myc breast 17.81 40 22.19 1.19
0.0E+00 4 BT-20 breast 17.9 38.73 20.83 -0.17 1.1E+00 5 BT-474
breast 17.65 40 22.35 1.35 0.0E+00 6 BT-483 breast 17.45 33.75 16.3
-4.7 2.6E+01 7 BT-549 breast 17.55 33.4 15.85 -5.15 3.6E+01 8
DU4475 breast 18.1 40 21.9 0.9 0.0E+00 9 H3396 breast 18.04 40
21.96 0.96 0.0E+00 10 HBL100 breast 17.02 40 22.98 1.98 0.0E+00 11
Her2 MCF-7 breast 19.26 40 20.74 -0.26 0.0E+00 12 HS 578T breast
17.83 36.64 18.81 -2.19 4.6E+00 13 MCF7 breast 17.83 40 22.17 1.17
0.0E+00 14 MCF-7/AdrR breast 17.23 36.44 19.21 -1.79 3.5E+00 18
MDAH 2774 breast 16.87 38.31 21.44 0.44 7.4E-01 16 MDA-MB-175-
breast 15.72 32.57 16.85 -4.15 1.8E+01 VII 17 MDA-MB-231 breast
17.62 40 22.38 1.38 0.0E+00 18 MDA-MB-453 breast 17.9 36.85 18.95
-2.05 4.1E+00 19 MDA-MB-468 breast 17.49 35.95 18.46 -2.54 5.8E+00
20 Pat-21 R60 breast 35.59 40 4.41 -16.59 ND 21 SKBR3 breast 17.12
35.66 18.54 -2.46 5.5E+00 22 T47D breast 18.86 36.2 17.34 -3.66
1.3E+01 23 UACC-812 breast 17.06 36.72 19.66 -1.34 2.5E+00 24
ZR-75-1 breast 15.95 40 24.05 3.05 0.0E+00 25 C-33A cervical 17.49
38.24 20.75 -0.25 1.2E+00 26 Ca Ski cervical 17.38 40 22.62 1.62
0.0E+00 27 HeLa cervical 17.59 40 22.41 1.41 0.0E+00 28 HT-3
cervical 17.42 36.52 19.1 -1.9 3.7E+00 29 ME-180 cervical 16.86
34.31 17.45 -3.55 1.2E+01 30 SiHa cervical 18.07 37.96 19.89 -1.11
2.2E+00 31 SW756 cervical 15.59 37.48 21.89 0.89 5.4E-01 32 CACO-2
colon 17.56 26.39 8.83 -12.17 4.6E+03 33 CCD-112Co colon 18.03
36.73 18.7 -2.3 4.9E+00 34 CCD-33Co colon 17.07 40 22.93 1.93
0.0E+00 35 Colo 205 colon 18.02 31.14 13.12 -7.88 2.4E+02 36 Colo
320DM colon 17.01 34.6 17.59 -3.41 1.1E+01 37 Colo201 colon 17.89
30.97 13.08 -7.92 2.4E+02 38 Cx-1 colon 18.79 34.05 15.26 -5.74
5.3E+01 39 ddH2O colon 40 40 0 -21 ND 40 HCT116 colon 17.59 35
17.41 -3.59 1.2E+01 41 HCT116/epo5 colon 17.71 36.42 18.71 -2.29
4.9E+00 42 HCT116/ras colon 17.18 33.03 15.85 -5.15 3.6E+01 43
HCT116/TX15C colon 17.36 31.3 13.94 -7.06 1.3E+02 R 44 HCT116/vivo
colon 17.7 34.26 16.56 -4.44 2.2E+01 45 HCT116/VM46 colon 17.87
35.07 17.2 -3.8 1.4E+01 46 HCT116/VP35 colon 17.3 33.35 16.05 -4.95
3.1E+01 47 HCT-8 colon 17.44 40 22.56 1.56 0.0E+00 48 HT-29 colon
17.9 33.29 15.39 -5.61 4.9E+01 49 LoVo colon 17.64 40 22.36 1.36
0.0E+00 50 LS 174T colon 17.93 36.1 18.17 -2.83 7.1E+00 51 LS123
colon 17.65 33.31 15.66 -5.34 4.1E+01 52 MIP colon 16.92 40 23.08
2.08 0.0E+00 53 SK-CO-1 colon 17.75 35.52 17.77 -3.23 9.4E+00 54
SW1417 colon 17.22 38.81 21.59 0.59 6.6E-01 55 SW403 colon 18.39
26.66 8.27 -12.73 6.8E+03 56 SW480 colon 17 37.82 20.82 -0.18
1.1E+00 57 SW620 colon 17.16 40 22.84 1.84 0.0E+00 58 SW837 colon
18.35 30.36 12.01 -8.99 5.1E+02 59 T84 colon 16.44 34.09 17.65
-3.35 1.0E+01 60 CCD-18Co colon, 17.19 38.1 20.91 -0.09 1.1E+00
fibroblast 61 HT-1080 fibrosarcoma 17.16 40 22.84 1.84 0.0E+00 62
CCRF-CEM leukemia 17.07 40 22.93 1.93 0.0E+00 63 HL-60 leukemia
17.54 40 22.46 1.46 0.0E+00 64 K562 leukemia 18.42 36.16 17.74
-3.26 9.6E+00 65 A-427 lung 18 40 22 1 0.0E+00 66 A549 lung 17.63
40 22.37 1.37 0.0E+00 67 Calu-3 lung 18.09 31.06 12.97 -8.03
2.6E+02 68 Calu-6 lung 16.62 36.23 19.61 -1.39 2.6E+00 69 ChaGo-K-1
lung 17.79 35.76 17.97 -3.03 8.2E+00 70 DMS 114 lung 18.14 37.86
19.72 -1.28 2.4E+00 71 LX-1 lung 18.17 36.99 18.82 -2.18 4.5E+00 72
MRC-5 lung 17.3 37.43 20.13 -0.87 1.8E+00 73 MSTO-211H lung 16.81
40 23.19 2.19 0.0E+00 74 NCI-H596 lung 17.73 34.14 16.41 -4.59
2.4E+01 75 SHP-77 lung 18.66 35.3 16.64 -4.36 2.1E+01 76 Sk-LU-1
lung 15.81 34.13 18.32 -2.68 6.4E+00 77 SK-MES-1 lung 17.1 40 22.9
1.9 0.0E+00 78 SW1271 lung 16.45 40 23.55 2.55 0.0E+00 79 SW1573
lung 17.14 37.06 19.92 -1.08 2.1E+00 80 SW900 lung 18.17 40 21.83
0.83 0.0E+00 81 Hs 294T melanoma 17.73 38.11 20.38 -0.62 1.5E+00 82
A2780/DDP-R ovarian 21.51 40 18.49 -2.51 0.0E+00 83 A2780/DDP-S
ovarian 17.89 39.67 21.78 0.78 5.8E-01 84 A2780/epo5 ovarian 17.54
35.29 17.75 -3.25 9.5E+00 85 A2780/TAX-R ovarian 18.4 37.65 19.25
-1.75 3.4E+00 86 A2780/TAX-S ovarian 17.83 36.54 18.71 -2.29
4.9E+00 87 Caov-3 ovarian 15.5 40 24.5 3.5 0.0E+00 88 ES-2 ovarian
17.22 37.13 19.91 -1.09 2.1E+00 89 HOC-76 ovarian 34.3 40 5.7 -15.3
ND 90 OVCAR-3 ovarian 17.09 40 22.91 1.91 0.0E+00 91 PA-1 ovarian
17.33 36.9 19.57 -1.43 2.7E+00 92 SW 626 ovarian 16.94 40 23.06
2.06 0.0E+00 93 UPN251 ovarian 17.69 36.52 18.83 -2.17 4.5E+00 94
LNCAP prostate 18.17 40 21.83 0.83 0.0E+00 95 PC-3 prostate 17.25
40 22.75 1.75 0.0E+00 96 A431 squamous 19.85 37.73 17.88 -3.12
8.7E+00
Example 5
Signal Transduction Assays
[0285] The activity of GPCRs or homologues thereof, can be measured
using any assay suitable for the measurement of the activity of a G
protein-coupled receptor, as commonly known in the art. Signal
transduction activity of a G protein-coupled receptor can be
monitor by monitoring intracellular Ca.sup.2+, cAMP, inositol
1,4,5-triphosphate (IP.sub.3), or 1,2-diacylglycerol (DAG). Assays
for the measurement of intracellular Ca.sup.2+ are described in
Sakurai et al. (EP 480 381). Intracellular IP.sub.3 ca be measured
using a kit available from Amersham, Inc. (Arlington Heights,
Ill.). A kit for measuring intracellular cAMP is available from
Diagnostic Products, Inc. (Los Angeles, Calif.).
[0286] Activation of a G protein-coupled receptor triggers the
release of Ca.sup.2+ ions sequestered in the mitochondria,
endoplasmic reticulum, and other cytoplasmic vesicles into the
cytoplasm. Fluorescent dyes, e.g., fura-2, can be used to measure
the concentration of free cytoplasmic Ca.sup.2+. The ester of
fura-2, which is lipophilic and can diffuse across the cell
membrane, is added to the media of the host cells expressing GPCRs.
Once inside the cell, the fura-2 ester is hydrolyzed by cytosolic
esterases to its non-lipophilic form, and then the dye cannot
diffuse back out of the cell. The non-lipophilic form of fura-2
will fluoresce when it binds to free Ca.sup.2+. The fluorescence
can be measured without lysing the cells at an excitation spectrum
of 340 nm or 380 nm and at fluorescence spectrum of 500 nm (Sakurai
et al., EP 480 381).
[0287] Upon activation of a G protein-coupled receptor, the rise of
free cytosolic Ca.sup.2+ concentrations is preceded by the
hydrolysis of phosphatidylinositol 4,5-bisphosphate. Hydrolysis of
this phospholipid by the phospholipase C yields 1,2-diacylglycerol
(DAG), which remains in the membrane, and water-soluble inositol
1,4,5-triphosphate (IP.sub.3). Binding of ligands or agonists will
increase the concentration of DAG and IP.sub.3. Thus, signal
transduction activity can be measured by monitoring the
concentration of these hydrolysis products.
[0288] To measure the IP.sub.3 concentrations, radioactivity
labeled .sup.3H-inositol is added to the media of host cells
expressing GPCRs. The .sup.3H-inositol is taken up by the cells and
incorporated into IP.sub.3. The resulting inositol triphosphate is
separated from the mono and di-phosphate forms and measured
(Sakurai et al., EP 480 381). Alternatively, Amersham provides an
inositol 1,4,5-triphosphate assay system. With this system Amersham
provides tritylated inositol 1,4,5-triphosphate and a receptor
capable of distinguishing the radioactive inositol from other
inositol phosphates. With these reagents an effective and accurate
competition assay can be performed to determine the inositol
triphosphate levels.
[0289] Cyclic AMP levels can be measured according to the methods
described in Gilman et al., Proc. Natl. Acad. Sci. 67:305-312
(1970). In addition, a kit for assaying levels of cAMP is available
from Diagnostic Products Corp. (Los Angeles, Calif.).
Example 5
GPCR Activity
[0290] Another method for screening compounds which are
antagonists, and thus inhibit activation of the receptor
polypeptide of the present invention is provided. This involves
determining inhibition of binding of labeled ligand, such as dATP,
dAMP, or UTP, to cells which have the receptor on the surface
thereof, or cell membranes containing the receptor. Such a method
further involves transfecting a eukaryotic cell with DNA encoding
the GPCR polypeptide such that the cell expresses the receptor on
its surface. The cell is then contacted with a potential antagonist
in the presence of a labeled form of a ligand, such as dATP, dAMP,
or UTP. The ligand can be labeled, e.g., by radioactivity,
fluorescence, or any detectable label commonly known in the art.
The amount of labeled ligand bound to the receptors is measured,
e.g., by measuring radioactivity associated with transfected cells
or membrane from these cells. If the compound binds to the
receptor, the binding of labeled ligand to the receptor is
inhibited as determined by a reduction of labeled ligand which
binds to the receptors. This method is called a binding assay.
Naturally, this same technique can be used to determine
agonists.
[0291] In a further screening procedure, mammalian cells, for
example, but not limited to, CHO, HEK 293, Xenopus Oocytes,
RBL-2H3, etc., which are transfected, are used to express the
receptor of interest. The cells are loaded with an indicator dye
that produces a fluorescent signal when bound to calcium, and the
cells are contacted with a test substance and a receptor agonist,
such as DATP, DAMP, or UTP. Any change in fluorescent signal is
measured over a defined period of time using, for example, a
fluorescence spectrophotometer or a fluorescence imaging plate
reader. A change in the fluorescence signal pattern generated by
the ligand indicates that a compound is a potential antagonist or
agonist for the receptor.
[0292] In yet another screening procedure, mammalian cells are
transfected to express the receptor of interest, and are also
transfected with a reporter gene construct that is coupled to
activation of the receptor (for example, but not limited to
luciferase or beta-galactosidase behind an appropriate promoter).
The cells are contacted with a test substance and the receptor
agonist (ligand), such as dATP, dAMP, or UTP, and the signal
produced by the reporter gene is measured after a defined period of
time. The signal can be measured using a luminometer,
spectrophotometer, fluorimeter, or other such instrument
appropriate for the specific reporter construct used. Inhibition of
the signal generated by the ligand indicates that a compound is a
potential antagonist for the receptor.
[0293] Another screening technique for antagonists or agonists
involves introducing RNA encoding the GPCR polypeptide into cells
(or CHO, HEK 293, RBL-2H3, etc.) to transiently or stably express
the receptor. The receptor cells are then contacted with the
receptor ligand, such as dATP, dAMP, or UTP, and a compound to be
screened. Inhibition or activation of the receptor is then
determined by detection of a signal, such as, cAMP, calcium,
proton, or other ions.
Example 6
Functional Characterization of HGPRBMY6
[0294] The putative GPCR HGPRBMY6 cDNA was PCR amplified using
PFU.TM. (Stratagene). The primers used in the PCR reaction were
specific to the HGPRBMY6 polynucleotide and were ordered from Gibco
BRL (5 prime primer: 5'-CGGGATGCCTAGATGCTTTCCTTTGCATTGTCACTTTC-3'
(SEQ ID NO:20). The following 3 prime primer was used to add a
Flag-tag epitope to the HGPRBMY2 polypeptide for
immunocytochemistry: 5'-CGGGGATCCCTACTTGTCGTCGTC-
GTCCTTGTAGTCCATGATGCTTTCCTT TGCATTGTCACTTTC-3'(SEQ ID NO:21). The
product from the PCR reaction was isolated from a 0.8% Agarose gel
(Invitrogen) and purified using a Gel Extraction Kit.TM. from
Qiagen.
[0295] The purified product was then digested overnight along with
the pcDNA3.1 Hygro.TM. mammalian expression vector from Invitrogen
using the HindIII and BamHI restriction enzymes (New England
Biolabs). These digested products were then purified using the Gel
Extraction Kit.TM. from Qiagen and subsequently ligated to the
pcDNA3.1 Hygro.TM. expression vector using a DNA molar ratio of 4
parts insert: 1 vector. All DNA modification enzymes were purchased
from NEB. The ligation was incubated overnight at 16 degrees
Celsius, after which time, one microliter of the mix was used to
transform DH5 alpha cloning efficiency competent E. coli.TM. (Gibco
BRL). A detailed description of the pcDNA3.1 Hygro.TM. mammalian
expression vector is available at the Invitrogen web site
(www.Invitrogen.com). The plasmid DNA from the ampicillin resistant
clones were isolated using the Wizard DNA Miniprep System.TM. from
Promega. Positive clones were then confirmed and scaled up for
purification using the Qiagen Maxiprep.TM. plasmid DNA purification
kit.
[0296] Cell Line Generation
[0297] The pcDNA3. lhygro vector containing the orphan HGPRBMY6
cDNA was used to transfect CHO-NFAT/CRE (Aurora Biosciences) cells
using Lipofectamine 2000.TM. according to the manufacturers
specifications (Gibco BRL). Two days later, the cells were split
1:3 into selective media (DMEM 11056, 600 .mu.g/ml Hygromycin, 200
.mu.g/ml Zeocin, 10% FBS). All cell culture reagents were purchased
from Gibco BRL-Invitrogen.
[0298] The CHO-NFAT/CRE and the CHO-NFAT G alpha 15 cell lines,
transiently or stably transfected with the orphan HGPRBMY6 GPCR,
were analyzed using the FACS Vantage SE.TM. (BD), fluorescence
microscopy (Nikon), and the LJL Analyst.TM. (Molecular Devices). In
this system, changes in real-time gene expression, as a consequence
of constitutive G-protein coupling of the orphan HGPRBMY6 GPCR, was
examined by analyzing the fluorescence emission of the transformed
cells at 447 nm and 518 nm. The changes in gene expression were
visualized using Beta-Lactamase as a reporter, that, when induced
by the appropriate signaling cascade, hydrolyzed an intracellularly
loaded, membrane-permeant ester substrate,
Cephalosporin-Coumarin-Fluorescein2/Acetoxymethyl (CCF2/AM.TM.
Aurora Biosciences; Zlokamik, et al., 1998). The CCF2/AM.TM.
substrate is a 7-hydroxycoumarin cephalosporin with a fluorescein
attached through a stable thioether linkage. Induced expression of
the Beta-Lactamase enzyme was readily apparent since each enzyme
molecule produced was capable of changing the fluorescence of many
CCF2/AM.TM. substrate molecules. A schematic of this cell based
system is shown below. 1
[0299] In summary, CCF2/AM.TM. is a membrane permeant,
intracellularly-trapped, fluorescent substrate with a cephalosporin
core that links a 7-hydroxycoumarin to a fluorescein. For the
intact molecule, excitation of the coumarin at 409 nm results in
Fluorescence Resonance Energy Transfer (FRET) to the fluorescein
which emits green light at 518 nm. Production of active
Beta-Lactamase results in cleavage of the Beta-Lactam ring, leading
to disruption of FRET, and excitation of the coumarin only--thus
giving rise to blue fluorescent emission at 447 nm.
[0300] Fluorescent emissions were detected using a Nikon-TE300
microscope equipped with an excitation filter (D405/10.times.-25),
dichroic reflector (430DCLP), and a barrier filter for dual
DAPI/FITC (510 nM) to visually capture changes in Beta-Lactamase
expression. The FACS Vantage SE was equipped with a Coherent
Enterprise II Argon Laser and a Coherent 302C Krypton laser. In
flow cytometry, UV excitation at 351-364 nm from the Argon Laser or
violet excitation at 407 nm from the Krypton laser were used. The
optical filters on the FACS Vantage SE were HQ460/50 m and HQ535/40
m bandpass separated by a 490 dichroic mirror.
[0301] Prior to analyzing the fluorescent emissions from the cell
lines as described above, the cells were loaded with the CCF2/AM
substrate. A 6.times.CCF2/AM loading buffer was prepared whereby 1
mM CCF2/AM (Aurora Biosciences) was dissolved in 100% DMSO (Sigma).
Stock solution (12 .mu.l) was added to 60 .mu.l of 100 mg/ml
Pluronic F127 (Sigma) in DMSO containing 0.1% Acetic Acid (Sigma).
This solution was added while vortexing to 1 mL of Sort Buffer (PBS
minus calcium and magnesium-Gibco-25 mM HEPES-Gibco- pH 7.4, 0.1%
BSA). Cells were placed in serum-free media and the 6.times.CCF2/AM
was added to a final concentration of 1.times.. The cells were then
loaded at room temperature for one to two hours, and then subjected
to fluorescent emission analysis as described herein. Additional
details relative to the cell loading methods and/or instrument
settings may be found by reference to the following publications:
see Zlokarnik, et al., 1998; Whitney et al., 1998; and BD
Biosciences, 1999.
[0302] Immunocytochemistry
[0303] The cell lines transfected and selected for expression of
Flag-epitope tagged orphan GPCRs were analyzed by
immunocytochemistry. The cells were plated at 1.times.10.sup.3 in
each well of a glass slide (VWR). The cells were rinsed with PBS
followed by acid fixation for 30 minutes at room temperature using
a mixture of 5% Glacial Acetic Acid/90% ethanol. The cells were
then blocked in 2% BSA and 0.1% Triton in PBS, and incubated for 2
h at room temperature or overnight at 4.degree. C. A monoclonal
anti-Flag FITC antibody was diluted at 1:50 in blocking solution
and incubated with the cells for 2 h at room temperature. Cells
were then washed three times with 0.1% Triton in PBS for five
minutes. The slides were overlayed with mounting media dropwise
with Biomedia--Gel Mount.TM. (Biomedia; Containing Anti-Quenching
Agent). Cells were examined at 10.times.magnification using the
Nikon TE300 equipped with FITC filter (535 nm).
[0304] There is strong evidence that certain GPCRs exhibit a cDNA
concentration-dependent constitutive activity through cAMP response
element (CRE) luciferase reporters (Chen et al., 1999). In an
effort to demonstrate functional coupling of HGPRBMY6 to known GPCR
second messenger pathways, the HGPRBMY6 polypeptide was expressed
at high constitutive levels in the CHO-NFAT/CRE cell line. To this
end, the HGPRBMY6 cDNA was PCR amplified and subdloned into the
pcDNA3.1 hygro.TM. mammalian expression vector as described herein.
Early passage CHO-NFAT/CRE cells were then transfected with the
resulting pcDNA3.1 hygro.TM./HGPRBMY6 construct. Transfected and
non-transfected CHO-NFAT/CRE cells (control) were loaded with the
CCF2 substrate and stimulated with 10 nM PMA, 1 .mu.M Thapsigargin
(NFAT stimulator), and 10 .mu.M Forskolin (CRE stimulator) to fully
activate the NFAT/CRE element. The cells were then analyzed for
fluorescent emission by FACS.
[0305] The FACS profile demonstrated the constitutive activity of
HGPRBMY6 in the CHO-NFAT/CRE line as evidenced by the significant
population of cells with blue fluorescent emission at 447 nm (see
FIG. 10: Blue Cells). FIG. 9 describes CHO-NFAT/CRE cell lines
transfected with the pcDNA3.1 Hygro.TM./HGPRBMY6 mammalian
expression vector. The cells were then analyzed via FACS according
to their wavelength emission at 518 nM (Channel R3--Green Cells),
and 447 nM (Channel R2--Blue Cells). As shown, overexpression of
HGPRBMY6 resulted in functional coupling and subsequent activation
of beta lactamase gene expression, as evidenced by the significant
number of cells with fluorescent emission at 447 nM relative to the
non-transfected control CHO-NFAT/CRE cells (shown in FIG. 10).
[0306] As expected, the NFAT/CRE response element in the
untransfected control cell line was not activated (i.e., beta
lactamase not induced), enabling the CCF2 substrate to remain
intact, and resulting in the green fluorescent emission at 518 nM
(see FIG. 9--Green Cells). FIG. 9 describes control CHO-NFAT/CRE
(Nuclear Factor Activator of Transcription (NFAT)/cAMP response
element (CRE)) cell lines, in the absence of the pcDNA3.1
Hygro.TM./HGPRBMY6 mammalian expression vector transfection. The
cells were analyzed via FACS (Fluorescent Assisted Cell Sorter)
according to their wavelength emission at 518 nM (Channel R3--Green
Cells), and 447 nM (Channel R2--Blue Cells). As shown, the vast
majority of cells emitted at 518 nM, with minimal emission observed
at 447 nM. The latter was expected since the NFAT/CRE response
elements remain dormant in the absence of an activated G-protein
dependent signal transduction pathway (e.g., pathways mediated by
Gq/11 or Gs coupled receptors). As a result, the cell permeant,
CCF2/AM.TM. (Aurora Biosciences; Zlokarnik, et al., 1998) substrate
remained intact and emitted light at 518 nM. A very low level of
leaky Beta Lactamase expression was detectable as evidenced by the
small population of cells emitting at 447 nm. Analysis of a stable
pool of cells transfected with HGPRBMY6 revealed constitutive
coupling of the cell population to the NFAT/CRE response element,
activation of Beta Lactamase and cleavage of the substrate (FIG.
10--Blue Cells). These results demonstrated that overexpression of
HGPRBMY6 leads to constitutive coupling of signaling pathways known
to be mediated by Gq/11 or G alpha 15/16 or Gs coupled receptors
that converge to activate either the NFAT or CRE response elements
respectively (Boss et al., 1996; Chen et al., 1999).
[0307] In an effort to further characterize the observed functional
coupling of the HGPRBMY6 polypeptide, its ability to couple to the
cAMP response element (CRE) independent of the NFAT response
element was examined. To this end, the HEK-CRE cell line that
contained only the integrated 3XCRE linked to the Beta-Lactamase
reporter was transfected with the pcDNA3.1 hygro.TM./HGPRBMY6
construct. Analysis of the fluorescence emission from this stable
pool showed that HGPRBMY6 constitutively coupled to the cAMP
mediated second messenger pathways (see FIG. 12 relative to FIG.
11). FIG. 11 describes HEK-CRE cell lines in the absence of the
pcDNA3.1 Hygro.TM./HGPRBMY6 mammalian expression vector
transfection. The cells were analyzed via FACS (Fluorescent
Assisted Cell Sorter) according to their wavelength emission at 518
nM (Channel R3-Green Cells), and 447 nM (Channel R2--Blue Cells).
As shown, the vast majority of cells emitted at 518 nM, with
minimal emission observed at 447 nM. The latter was expected since
the CRE response elements remain dormant in the absence of an
activated G-protein dependent signal transduction pathway (e.g.,
pathways mediated by Gs coupled receptors). As a result, the cell
permeant, CCF2/AM.TM. (Aurora Biosciences; Zlokamik, et al., 1998)
substrate remained intact and emitted light at 518 nM. FIG. 12
describes HEK-CRE cell lines transfected with the pcDNA3.1
Hygro.TM./HGPRBMY6 mammalian expression vector analyzed via FACS
according to their wavelength emission at 518 nM (Channel R3--Green
Cells), and 447 nM (Channel R2--Blue Cells). As shown,
overexpression of HGPRBMY6 in the HEK-CRE cells resulted in
functional coupling, as evidenced by the insignificant background
level of cells with fluorescent emission at 447 nM. Experiments
have shown that known Gs coupled receptors demonstrate constitutive
activation when overexpressed in the HEK-CRE cell line. For
example, direct activation of adenylate cyclase using 10 .mu.M
Forskolin has been shown to activate CRE and the subsequent
induction of Beta-Lactamase in the HEK-CRE cell line (data not
shown). In conclusion, the results were consistent with HGPRBMY6
representing a functional GPCR analogous to known Gs coupled
receptors (Boss et al., 1996).
[0308] In an effort to further characterize the observed functional
coupling of the HGPRBMY6 polypeptide, its ability to couple to a G
protein was examined. To this end, the promiscuous G protein, G
alpha 15 was utilized. Specific domains of alpha subunits of G
proteins have been shown to control coupling to GPCRs (Blahos et
al., 2001). It has also been demonstrated that the extreme
C-terminal 20 amino acids of either G alpha 15 or 16 confer the
unique ability of these G proteins to couple to many GPCRs,
including those that naturally do not stimulate PLC (Blahos et al.,
2001). Indeed, both G alpha 15 and 16 were shown to couple a wide
variety of GPCRs to Phospholipase C activation of calcium mediated
signaling pathways (including the NFAT-signaling pathway)
(Offermanns & Simon). To demonstrate that HGPRBMY6 was
functioning as a GPCR, the CHO-NFAT G alpha 15 cell line that
contained only the integrated NFAT response element linked to the
Beta-Lactamase reporter was transfected with the pcDNA3.1
hygro.TM./HGPRBMY6 construct. Analysis of the fluorescence emission
from this stable pool showed that HGPRBMY6 constitutively coupled
to the NFAT mediated second messenger pathways via G alpha 15 (see
FIGS. 13 and 14). FIG. 13 describes control CHO-NFAT G alpha 15
(Nuclear Factor Activator of Transcription (NFAT)) cell lines, in
the absence of the pcDNA3.1 Hygro.TM./HGPRBMY6 mammalian expression
vector transfection. The cells were analyzed via FACS (Fluorescent
Assisted Cell Sorter) according to their wavelength emission at 518
nM (Channel R3--Green Cells), and 447 nM (Channel R2--Blue Cells).
As shown, the vast majority of cells emitted at 518 nM, with
minimal emission observed at 447 nM. The latter was expected since
the NFAT response elements remained dormant in the absence of an
activated G-protein dependent signal transduction pathway (e.g.,
pathways mediated by G alpha 15 Gq/11 or Gs coupled receptors). As
a result, the cell permeant, CCF2/AM.TM. (Aurora Biosciences;
Zlokamik, et al., 1998) substrate remained intact and emitted light
at 518 nM. FIG. 14 describes CHO-NFAT G alpha 15 cell lines
transfected with the pcDNA3.1 Hygro .TM./HGPRBMY6 mammalian
expression vector. The cells were analyzed and sorted via FACS
according to their wavelength emission at 518 nM (Channel R3--Green
Cells), and 447 nM (Channel R2--Blue Cells). As shown,
overexpression of HGPRBMY6 resulted in functional coupling and
subsequent activation of beta lactamase gene expression, as
evidenced by the significant number of cells with fluorescent
emission at 447 nM relative to the non-transfected control CHO-NFAT
G alpha 15 cells (shown in FIG. 13).
[0309] In conclusion, the results were consistent with HGPRBMY6
representing a functional GPCR analogous to known G alpha 15
coupled receptors. Therefore, constitutive expression of HGPRBMY6
in the CHO-NFAT G alpha 15 cell line leads to NFAT activation
through accumulation of intracellular Ca.sup.2+ as has been
demonstrated for the M3 muscarinic receptor (Boss et al.,
1996).
[0310] Demonstration of Cellular Expression
[0311] HGPRBMY6 was tagged at the C-terminus using the Flag epitope
and inserted into the pcDNA3.1 hygro.TM. expression vector, as
described herein. Immunocytochemistry of CHO-NFAT G alpha 15 cell
lines transfected with the Flag-tagged HGPRBMY6 construct with FITC
conjugated monoclonal antibody directed against FLAG demonstrated
that HGPRBMY6 is indeed a cell surface receptor. The
immunocytochemistry also confirmed expression of the HGPRBMY6 in
the CHO-NFAT G alpha 15 cell lines. Briefly, CHO-NFAT G alpha 15
cell lines were transfected with pcDNA3.1 hygro.TM./HGPRBMY6-Flag
vector, fixed with 70% methanol, and permeablized with 0.1%
TritonX100. The cells were then blocked with 1% Serum and incubated
with a FITC conjugated Anti Flag monoclonal antibody at 1:50
dilution in PBS-Triton. The cells were then washed several times
with PBS-Triton, overlayed with mounting solution, and fluorescent
images were captured (see FIG. 15). FIG. 15 describes CHO-NFAT/CRE
cell lines transfected with the pcDNA 3.1 Hygro.TM./HGPRBMY6-FLAG
mammalian expression vector subjected to immunocytochemistry using
an FITC conjugated monoclonal antibody against FLAG. Panel A shows
the transfected CHO-NFAT/CRE cells under visual wavelengths, and
panel B shows the clearly evident fluorescent emission that is
consistent with the HGPRBMY6 polypeptide representing a member of
the GPCR family. The control cell line, non-transfected CHO-NFAT G
alpha 15 cell line, exhibited no detectable background fluorescence
(FIG. 15). The HGPRBMY6-FLAG tagged expressing CHO-NFAT G alpha 15
line exhibited specific plasma membrane expression as indicated
(FIG. 15).
[0312] These data provided clear evidence that HGPRBMY6 was
expressed in these cells and the majority of the protein was
localized to the cell surface. Cell surface localization was
consistent with HGPRBMY6 representing a 7 transmembrane domain
containing GPCR. Taken together, the data indicated that HGPRBMY6
was a cell surface GPCR that can function through increases in
either cAMP or Ca.sup.2+ signal transduction pathways via G alpha
15.
[0313] Screening Paradigm
[0314] The Aurora Beta-Lactamase technology provided a clear path
for identifying agonists and antagonists of the HGPRBMY6
polypeptide. Cell lines that exhibited a range of constitutive
coupling activity were identified by sorting through HGPRBMY6
transfected cell lines using the FACS Vantage SE (see FIG. 16). For
example, cell lines were sorted that had an intermediate level of
orphan GPCR expression, which also correlated with an intermediate
coupling response, using the LJL analyst. Such cell lines provided
the opportunity to screen, indirectly, for both agonists and
antogonists of HGPRBMY6 by searching for inhibitors that block the
beta lactamase response, or agonists that increase the beta
lactamase response. As described herein, modulating the expression
level of beta lactamase directly correlated with the level of
cleaved CCF2 substrate. For example, this screening paradigm has
been shown to work for the identification of modulators of a known
GPCR, 5HT6, that couples through Adenylate Cyclase, in addition to,
the identification of modulators of the 5HT2c GPCR, that couples
through changes in [Ca.sup.2+]i. The data shown represent cell
lines that have been engineered with the desired pattern of
HGPRBMY6 expression to enable the identification of potent small
molecule agonists and antagonists. FIG. 16 describes several
CHO-NFAT/CRE cell lines transfected with the pcDNA3.1
Hygro.TM./HGPRBMY6 mammalian expression vector isolated via FACS
that had either intermediate or high beta lactamase expression
levels of constitutive activation. Panel A shows untransfected
CHO-NFAT/CRE cells prior to stimulation with 10 nM PMA, 1 .mu.M
Thapsigargin, and 10 .mu.M Forskolin (-P/T/F) that are
representative of the relative background level of beta lactamase
expression. Panel B shows CHO-NFAT/CRE cells after stimulation with
10 nM PMA, 1 .mu.M Thapsigargin, and 10 .mu.M Forskolin (+P/T/F),
where the cells filly activated the CRE-NFTA response element
demonstrating the dynamic range of the assay. Panel C shows a
representative orphan GPCR (oGPCR) transfected in CHO-NFAT/CRE
cells that had an intermediate level of beta lactamase expression,
while panel D shows a representative orphan GPCR transfected in a
CHO-NFAT/CRE cell line that had a high level of constitutive beta
lactamase expression. HGPRBMY6 modulator screens may be carried out
using a variety of high throughput methods known in the art, though
preferably using the fully automated Aurora UHTSS system. (FIG. 16;
panel a).
Example 7
Phage Display Methods for Identifying Peptide Ligands or Modulators
of Orphan GPCRs
[0315] Library Construction
[0316] Two HGPRBMY libraries were used for identifying peptides
that may function as modulators. Specifically, a 15-mer library was
used to identify peptides that may function as agonists or
antagonists. The 15-mer library is an aliquot of the 15-mer library
originally constructed by G. P. Smith (Scott, J K and Smith, G P.
1990, Science 249:386-390). A 40-mer library was used for
identifying natural ligands and constructed essentially as
previously described (B K Kay, et al. 1993, Gene 128:59-65), with
the exception that a 15 base pair complementary region was used to
anneal the two oligonucleotides, as opposed to 6, 9, or 12 base
pairs, as described below.
[0317] The oligos used were: Oligo 1: 5'-CGAAGCGTAAGGGCCCAGCCGGCC
(NNK.times.20) CCGGGTCCGGGCGGC-3' (SEQ ID NO:67) and Oligo2:
5'-AAAAGGAAAAAAGCGGCCGC (VNN.times.20) GCCGCCCGGACCCGG-3' (SEQ ID
NO:68), where N=A+G+C+T and K=C+G+T and V=C+A+G.
[0318] The oligos were annealed through their 15 base pair
complimentary sequences which encode a constant ProGlyProGlyGly
(SEQ ID NO:69) pentapeptide sequence between the random 20 amino
acid segments, and then extended by standard procedure using Klenow
enzyme. This was followed by endonuclease digestion using Sfi1 and
Not1 enzymes and ligation to Sfi1 and Not1 cleaved pCantab5E
(Pharnacia). The ligation mixture was electroporated into E. coli
XL1Blue and phage clones were essentially generated as suggested by
the manufacturer for making ScFv antibody libraries in
pCantab5E.
[0319] Sequencing Bound Phage
[0320] Standard procedures commonly known in the art were used.
Phage in eluates were infected into E. coli host strain (TG1 for
the 15-mer library; XL1Blue for the 40-mer library) and plated for
single colonies. Colonies were grown in liquid and sequenced by
standard procedure which involved: 1) generating PCR product with
suitable primers of the library segments in the phage genome (15
mer library) or pCantab5E (40 mer library); and 2) sequencing PCR
products using one primer of each PCR primer pair. Sequences were
visually inspected or by using the Vector NTI alignment tool.
[0321] Peptide Modulators Of The Present Invention
[0322] The following serve as non-limiting examples of
peptides:
[0323] FAGQIIWYDALDTLM (SEQ ID NO:70)
[0324] SDFVGGFWFWDSLFN (SEQ ID NO:71)
[0325] GDFWYEACESSCAFW (SEQ ID NO:72)
[0326] LEWGSDVFYDVYDCC (SEQ ID NO:73)
[0327] RIDSCAKYFLRSCD (SEQ ID NO:74)
[0328] CLRSGTGCAFQLYRF (SEQ ID NO:75)
[0329] FRVSRVWNPPSFDSA (SEQ ID NO:76)
[0330] HAYVECNDTDCRVWF (SEQ ID NO:77)
[0331] Peptide Synthesis
[0332] Peptides were synthesized on Fmoc-Knorr amide resin
[N-(9-fluorenyl)methoxycarbonyl-Knorr amide-resin; Midwest Biotech;
Fishers, Ind.] with an Applied Biosystems (Foster City, Calif.)
model 433A synthesizer and the FastMoc chemistry protocol (0.25
mmol scale) supplied with the instrument. Amino acids were double
coupled as their N-.alpha.-Fmoc-derivatives and reactive side
chains were protected as follows: Asp, Glu: t-Butyl ester (OtBu);
Ser, Thr, Tyr: t-Butyl ether (tBu); Asn, Cys, Gln, His:
Triphenylmethyl (Trt); Lys, Trp: t-Butyloxycarbonyl (Boc); Arg:
2,2,4,6,7-Pentamethyldihydrobenzofuran-5-s- ulfonyl (Pbf). After
the final double coupling cycle, the N-terminal Fmoc group was
removed by the multi-step treatment with piperidine in
N-Methylpyrrolidone described by the manufacturer. The N-terminal
free amines were then treated with 10% acetic anhydride, 5%
Diisopropylamine in N-Methylpyrrolidone to yield the
N-acetyl-derivative. The protected peptidyl-resins were
simultaneously deprotected and removed from the resin by standard
methods. The lyophilized peptides were purified on C.sub.18 to
apparent homogeneity as judged by RP-HPLC analysis. Predicted
peptide molecular weights were verified by electrospray mass
spectrometry (J. Biol. Chem. 273:12041-12046, 1998).
[0333] Cyclic analogs were prepared from the crude linear products.
The cysteine disulfide was formed using one of the following
methods:
[0334] Method 1
[0335] A sample of the crude peptide was dissolved in water at a
concentration of 0.5 mg/mL and the pH adjusted to 8.5 with
NH.sub.4OH. The reaction was stirred at room temperature, and
monitored by RP-HPLC. Once completed, the reaction was adjusted to
pH 4 with acetic acid and lyophilized. The product was purified and
characterized as above.
[0336] Method 2
[0337] A sample of the crude peptide was dissolved at a
concentration of 0.5 mg/mL in 5% acetic acid. The pH was adjusted
to 6.0 with NH.sub.4OH. DMSO (20% by volume) was added and the
reaction was stirred overnight. After analytical RP-HPLC analysis,
the reaction was diluted with water and triple lyophilized to
remove DMSO. The crude product was purified by preparative RP-HPLC
(JACS. 113:6657, 1991)
[0338] Assessing Affect of Peptides on GPCR Function
[0339] The effect of any one of these peptides on the function of
the GPCR of the present invention may be determined by adding an
effective amount of each peptide to each functional assay.
Representative functional assays are described more specifically
herein, particularly Example 6.
[0340] Uses Of The Peptide Modulators Of The Present Invention
[0341] The aforementioned peptides of the present invention are
useful for a variety of purposes, though most notably for
modulating the function of the GPCR of the present invention, and
potentially with other GPCRs of the same G-protein coupled receptor
subclass (e.g., peptide receptors, adrenergic receptors, purinergic
receptors, etc.), and/or other subclasses known in the art. For
example, the peptide modulators of the present invention may be
useful as HGPRBMY6 agonists. Alternatively, the peptide modulators
of the present invention may be useful as HGPRBMY6 antagonists of
the present invention. In addition, the peptide modulators of the
present invention may be useful as competitive inhibitors of the
HGPRBMY6 cognate ligand(s), or may be useful as non-competitive
inhibitors of the HGPRBMY6 cognate ligand(s).
[0342] Furthermore, the peptide modulators of the present invention
may be useful in assays designed to either deorphan the HGPRBMY6
polypeptide of the present invention, or to identify other agonists
or antagonists of the HGPRBMY6 polypeptide of the present
invention, particularly small molecule modulators.
Example 8
Method of Creating N- and C-terminal Deletion Mutants Corresponding
to the HGPRBMY6 Polypeptide
[0343] As described elsewhere herein, the present invention
encompasses the creation of N- and C-terminal deletion mutants, in
addition to any combination of N- and C-terminal deletions thereof,
corresponding to the HGPRBMY6 polypeptide of the present invention.
A number of methods are available to one skilled in the art for
creating such mutants. Such methods may include a combination of
PCR amplification and gene cloning methodology. Although one of
skill in the art of molecular biology, through the use of the
teachings provided or referenced herein, and/or otherwise known in
the art as standard methods, could readily create each deletion
mutants of the present invention, exemplary methods are described
below.
[0344] Briefly, using the isolated cDNA clone encoding the
full-length HGPRBMY6 polypeptide sequence, appropriate primers of
about 15-25 nucleotides derived from the desired 5' and 3'
positions of SEQ ID NO:1 may be designed to PCR amplify, and
subsequently clone, the intended N- and/or C-terminal deletion
mutant. Such primers could comprise, for example, an inititation
and stop codon for the 5' and 3' primer, respectively. Such primers
may also comprise restriction sites to facilitate cloning of the
deletion mutant post amplification. Moreover, the primers may
comprise additional sequences, such as, for example, flag-tag
sequences, kozac sequences, or other sequences discussed and/or
referenced herein.
[0345] For example, in the case of the D198 to I560 N-terminal
deletion mutant, the following primers could be used to amplify a
cDNA fragment corresponding to this deletion mutant:
2 5' 5'-GCAGCA GCGGCCGC GACATATTATCCAACGTTGGATGTG-3' (SEQ ID NO:78)
Primer NotI 3' 5'-GCAGCA GTCGAC GATGCTTTCCTTTGCATTGTCAC-3' (SEQ ID
NO:79) Primer SalI
[0346] For example, in the case of the M1 to Y483 C-terminal
deletion mutant, the following primers could be used to amplify a
cDNA fragment corresponding to this deletion mutant:
3 5' 5'-GCAGCA GCGGCCGC ATGGAGACTTATTCCTTGTCTTTGG-3' (SEQ ID NO:80)
Primer NotI 3' 5'-GCAGCA GTCGAC GTACAGGATAAAAATTTGCAATCCC-3' (SEQ
ID NO:81) Primer SalI
[0347] Representative PCR amplification conditions are provided
below, although the skilled artisan would appreciate that other
conditions may be required for efficient amplification. A 100 ul
PCR reaction mixture may be prepared using 10 ng of the template
DNA (cDNA clone of HGPRBMY6), 200 uM 4 dNTPs, 1 uM primers, 0.25 U
Taq DNA polymerase (PE), and standard Taq DNA polymerase buffer.
Typical PCR cycling condition are as follows:
4 20-25 cycles: 45 sec, 93 degrees 2 min, 50 degrees 2 min, 72
degrees 1 cycle: 10 min, 72 degrees
[0348] After the final extension step of PCR, 5 U Klenow Fragment
may be added and incubated for 15 min at 30 degrees.
[0349] Upon digestion of the fragment with the NotI and SalI
restriction enzymes, the fragment could be cloned into an
appropriate expression and/or cloning vector which has been
similarly digested (e.g., pSport1, among others). The skilled
artisan would appreciate that other plasmids could be equally
substituted, and may be desirable in certain circumstances. The
digested fragment and vector are then ligated using a DNA ligase,
and then used to transform competent E.coli cells using methods
provided herein and/or otherwise known in the art.
[0350] The 5' primer sequence for amplifying any additional
N-terminal deletion mutants may be determined by reference to the
following formula:
(S+(X*3)) to ((S+(X*3))+25),
[0351] wherein `S` is equal to the nucleotide position of the
initiating start codon of the HGPRBMY6 gene (SEQ ID NO:1), and `X`
is equal to the most N-terminal amino acid of the intended
N-terminal deletion mutant. The first term will provide the start
5' nucleotide position of the 5' primer, while the second term will
provide the end 3' nucleotide position of the 5' primer
corresponding to sense strand of SEQ ID NO:1. Once the
corresponding nucleotide positions of the primer are determined,
the final nucleotide sequence may be created by the addition of
applicable restriction site sequences to the 5' end of the
sequence, for example. As referenced herein, the addition of other
sequences to the 5' primer may be desired in certain circumstances
(e.g., kozac sequences, etc.).
[0352] The 3' primer sequence for amplifying any additional
N-terminal deletion mutants may be determined by reference to the
following formula:
(S+(X*3)) to ((S+(X*3))-25),
[0353] wherein `S` is equal to the nucleotide position of the
initiating start codon of the HGPRBMY6 gene (SEQ ID NO:1), and `X`
is equal to the most C-terminal amino acid of the intended
N-terminal deletion mutant. The first term will provide the start
5' nucleotide position of the 3' primer, while the second term will
provide the end 3' nucleotide position of the 3' primer
corresponding to the anti-sense strand of SEQ ID NO:1. Once the
corresponding nucleotide positions of the primer are determined,
the final nucleotide sequence may be created by the addition of
applicable restriction site sequences to the 5' end of the
sequence, for example. As referenced herein, the addition of other
sequences to the 3' primer may be desired in certain circumstances
(e.g., stop codon sequences, etc.). The skilled artisan would
appreciate that modifications of the above nucleotide positions may
be necessary for optimizing PCR amplification.
[0354] The same general formulas provided above may be used in
identifying the 5' and 3' primer sequences for amplifying any
C-terminal deletion mutant of the present invention. Moreover, the
same general formulas provided above may be used in identifying the
5' and 3' primer sequences for amplifying any combination of
N-terminal and C-terminal deletion mutant of the present invention.
The skilled artisan would appreciate that modifications of the
above nucleotide positions may be necessary for optimizing PCR
amplification.
[0355] In preferred embodiments, the following N-terminal HGPRBMY6
deletion polypeptides are encompassed by the present invention:
M1-I560, E2-I560, T3-I560, Y4-I560, S5-I560, L6-I560, S7-I560,
L8-I560, G9-I560, N10-I560, Q11-I560, S12-I560, V13-I560, V14-I560,
E15-I560, P16-I560, N17-I560, I18-I560, A19-I560, I20-I560,
Q21-I560, S22-I560, A23-I560, N24-I560, F25-I560, S26-I560,
S27-I560, E28-I560, N29-I560, A30-I560, V31-I560, G32-I560,
P33-I560, S34-I560, N35-I560, V36-I560, R37-I560, F38-I560,
S39-I560, V40-I560, Q41-I560, K42-I560, G43-I560, A44-I560,
S45-I560, S46-I560, S47-I560, L48-I560, V49-I560, S50-I560,
S51-I560, S52-I560, T53-I560, F54-I560, I55-I560, H56-I560,
T57-I560, N58-I560, V59-I560, D60-I560, G61-I560, L62-I560,
N63-I560, P64-I560, D65-I560, A66-I560, Q67-I560, T68-I560,
E69-I560, L70-I560, Q71-I560, V72-I560, L73-I560, L74-I560,
N75-I560, M76-I560, T77-I560, K78-I560, N79-I560, Y80-I560,
T81-I560, K82-I560, T83-I560, C84-I560, G85-I560, F86-I560,
V87-I560, V88-I560, Y89-I560, Q90-I560, N91-I560, D92-I560,
K93-I560, L94-I560, F95-I560, Q96-I560, S97-I560, K98-I560,
T99-I560, F100-I560, T101-I560, A102-I560, K103-I560, S104-I560,
D105-I560, F106-I560, S107-I560, Q108-I560, K109-I560, I110-I560,
I111-I560, S112-I560, S113-I560, K114-I560, T115-I560, D116-I560,
E117-I560, N118-I560, E119-I560, Q120-I560, D121-I560, Q122-I560,
S123-I560, A124-I560, S125-I560, V126-I560, D127-I560, M128-I560,
V129-I560, F130-I560, S131-I560, P132-I560, K133-I560, Y134-I560,
N135-I560, Q136-I560, K137-I560, E138-I560, F139-I560, Q140-I560,
L141-I560, Y142-I560, S14-I560, Y144-I560, A145-I560, C146-I560,
V147-I560, Y148-I560, W149-I560, N150-I560, L151-I560, S152-I560,
A153-I560, K154-I560, D155-I560, W156-I560, D157-I560, T158-I560,
Y159-I560, G160-I560, C161-I560, Q162-I560, K163-I560, D164-I560,
K165-I560, G166-I560, T167-I560, D168-I560, G169-I560, F170-I560,
L171-I560, R172-I560, C173-I560, R174-I560, C175-I560, N176-I560,
H177-I560, T178-I560, T179-I560, N180-I560, F181-I560, A182-I560,
V183-I560, L184-I560, M185-I560, T186-I560, F187-I560, K188-I560,
K189-I560, D190-I560, Y191-I560, Q192-I560, Y193-I560, P194-I560,
K195-I560, S196-I560, L197-I560, D198-I560, I199-I560, L200-I560,
S201-I560, N202-I560, V203-I560, G204-I560, C205-I560, A206-I560,
L207-I560, S208-I560, V209-I560, T210-I560, G211-I560, L212-I560,
A213-I560, L214-I560, T215-I560, V216-I560, I217-I560, F218-I560,
Q219-I560, I220-I560, V221-I560, T222-I560, R223-I560, K224-I560,
V225-I560, R226-I560, K227-I560, T228-I560, S229-I560, V230-I560,
T231-I560, W232-I560, V233-I560, L234-I560, V235-I560, N236-I560,
L237-I560, C238-I560, 1239-I560, S240-I560, M241-I560, L242-I560,
I243-I560, F244-I560, N245-I560, L246-I560, L247-I560, F248-I560,
V249-I560, F250-I560, G251-I560, 1252-I560, E253-I560, N254-I560,
S255-I560, N256-I560, K257-I560, N258-I560, L259-I560, Q260-I560,
T261-I560, S262-I560, D263-I560, G264-I560, D265-I560, I266-I560,
N267-I560, N268-I560, I269-I560, D270-I560, F271-I560, D272-I560,
N273-I560, N274-I560, D275-I560, I276-I560, P277-I560, R278-I560,
T279-I560, D280-I560, T281-I560, I282-I560, N283-I560, I284-I560,
P285-I560, N286-I560, P287-I560, M288-I560, C289-I560, T290-I560,
A291-I560, I292-I560, A293-I560, A294-I560, L295-I560, L296-I560,
H297-I560, Y298-I560, F299-I560, L300-I560, L301-I560, V302-I560,
T303-I560, F304-I560, T305-I560, W306-I560, N307-I560, A308-I560,
L309-I560, S310-I560, A311-I560, A312-I560, Q313-I560, L314-I560,
Y315-I560, Y316-I560, L317-I560, L318-I560, I319-I560, R320-I560,
T321-I560, M322-I560, K323-I560, P324-I560, L325-I560, P326-I560,
R327-I560, H328-I560, F329-I560, I330-I560, L331-I560, F332-I560,
I333-I560, S334-I560, L335-I560, I336-I560, G337-I560, W338-I560,
G339-I560, V340-I560, P341-I560, A342-I560, I343-I560, V344-I560,
V345-I560, A346-I560, I347-I560, T348-I560, V349-I560, G350-I560,
V351-I560, I352-I560, Y353-I560, S354-I560, Q355-I560, N356-I560,
G357-I560, N358-I560, N359-I560, P360-I560, Q361-I560, W362-I560,
E363-I560, L364-I560, D365-I560, Y366-I560, R367-I560, Q368-I560,
E369-I560, K370-I560, I371-I560, C372-I560, W373-I560, L374-I560,
A375-I560, I376-I560, P377-I560, E378-I560, P379-I560, N380-I560,
G381-I560, V382-I560, I383-I560, K384-I560, S385-I560, P386-I560,
L387-I560, L388-I560, W389-I560, S390-I560, F391-I560, I392-I560,
V393-I560, P394-I560, V395-I560, T396-I560, I397-I560, I398-I560,
L399-I560, I400-I560, S401-I560, N402-I560, V403-I560, V404-I560,
M405-I560, F406-I560, I407-I560, T408-I560, I409-I560, S410-I560,
I411-I560, K412-I560, V413-I560, L414-I560, W415-I560, K416-I560,
N417-I560, N418-I560, Q419-I560, N420-I560, L421-I560, T422-I560,
S423-I560, T424-I560, K425-I560, K426-I560, V427-I560, S428-I560,
S429-I560, M430-I560, K431-I560, K432-I560, I433-I560, V434-I560,
S435-I560, T436-I560, L437-I560, S438-I560, V439-I560, A440-I560,
V441-I560, V442-I560, F443-I560, G444-I560, I445-I560, T446-I560,
W447-I560, I448-I560, L449-I560, A450-I560, Y451-I560, L452-I560,
M453-I560, L454-I560, V455-I560, N456-I560, D457-I560, D458-I560,
S459-I560, I460-I560, R461-I560, I462-I560, V463-I560, F464-I560,
S465-I560, Y466-I560, I467-I560, F468-I560, C469-I560, L470-I560,
F471-I560, N472-I560, T473-I560, T474-I560, Q475-I560, G476-I560,
L477-I560, Q478-I560, I479-I560, F480-I560, I481-I560, L482-I560,
Y483-I560, T484-I560, V485-I560, R486-I560, T487-I560, K488-I560,
V489-I560, F490-I560, Q491-I560, S492-I560, E493-I560, A494-I560,
S495-I560, K496-I560, V497-I560, L498-I560, M499-I560, L500-I560,
L501-I560, S502-I560, S503-I560, I504-I560, G505-I560, R506-I560,
R507-I560, K508-I560, S509-I560, L510-I560, P511-I560, S512-I560,
V513-I560, T514-I560, R515-I560, P516-I560, R517-I560, L518-I560,
R519-I560, V520-I560, K521-I560, M522-I560, Y523-I560, N524-I560,
F525-I560, L526-I560, R527-I560, S528-I560, L529-I560, P530-I560,
T531-I560, L532-I560, H533-I560, E534-I560, R535-I560, F536-I560,
R537-I560, L538-I560, L539-I560, E540-I560, T541-I560, S542-I560,
P543-I560, S544-I560, T545-I560, E546-I560, E547-I560, I548-I560,
T549-I560, L550-I560, S551-I560, E552-I560, S553-I560, and/or
D554-I560 of SEQ ID NO:2. Polynucleotide sequences encoding these
polypeptides are also included in SEQ ID NO:1. The present
invention also encompasses the use of these N-terminal HGPRBMY6
deletion polypeptides as immunogenic and/or antigenic epitopes as
described elsewhere herein.
[0356] In preferred embodiments, the following C-terminal HGPRBMY6
deletion polypeptides are encompassed by the present invention:
M1-I560, M1-S559, M1-E558, M1-K557, M1-A556, M1-N555, M1-D554,
M1-S553, M1-E552, M1-S551, M1-L550, M1-T549, M1-I548, M1-E547,
M1-E546, M1-T545, M1-S544, M1-P543, M1-S542, M1-T541, M1-E540,
M1-L539, M1-L538, M1-R537, M1-F536, M1-R535, M1-E534, M1-H533,
M1-L532, M1-T531, M1-P530, M1-L529, M1-S528, M1-R527, M1-L526,
M1-F525, M1-N524, M1-Y523, M1-M522, M1-K521, M1-V520, M1-R519,
M1-L518, M1-R517, M1-P516, M1-R515, M1-T514, M1-V513, M1-S512,
M1-P511, M1-L510, M1-S509, M1-K508, M1-R507, M1-R506, M1-G505,
M1-I504, M1-S503, M1-S502, M1-L501, M1-L500, M1-M499, M1-L498,
M1-V497, M1-K496, M1-S495, M1-A494, M1-E493, M1-S492, M1-Q491,
M1-F490, M1-V489, M1-K488, M1-T487, M1-R486, M1-V485, M1-T484,
M1-Y483, M1-L482, M1-I481, M1-F480, M1-I479, M1-Q478, M1-L477,
M1-G476, M1-Q475, M1-T474, M1-T473, M1-N472, M1-F471, M1-L470,
M1-C469, M1-F468, M1-I467, M1-Y466, M1-S465, M1-F464, M1-V463,
M1-I472, M1-R461, M1-I460, M1-S459, M1-D458, M1-D457, M1-N456,
M1-V455, M1-L454, M1-M453, M1-L452, M1-Y451, M1-A450, M1-L449,
M1-I448, M1-W447, M1-T446, M1-I445, M1-G444, M1-F443, M1-V442,
M1-V441, M1-A440, M1-V439, M1-S438, M1-L437, M1-T436, M1-S435,
M1-V434, M1-I433, M1-K432, M1-K431, M1-M430, M1-S429, M1-S428,
M1-V427, M1-K426, M1-K425, M1-T424, M1-S423, M1-T422, M1-L421,
M1-N420, M1-Q419, M1-N418, M1-N417, M1-K416, M1-W415, M1-L414,
M1-V413, M1-K412, M1-I411, M1-S410, M1-I409, M1-T408, M1-I407,
M1-F406, M1-M405, M1-V404, M1-V403, M1-N402, M1-S401, M1-I400,
M1-L399, M1-I398, M1-I397, M1-T396, M1-V395, M1-P394, M1-V393,
M1-I392, M1-F391, M1-S390, M1-W389, M1-L388, M1-L387, M1-P386,
M1-S385, M1-K384, M1-I383, M1-V382, M1-G381, M1-N380, M1-P379,
M1-E378, M1-P377, M1-I376, M1-A375, M1-L374, M1-W373, M1-C372,
M1-I371, M1-K370, M1-E369, M1-Q368, M1-R367, M1-Y366, M1-D365,
M1-L364, M1-E363, M1-W362, M1-Q361, M1-P360, M1-N359, M1-N358,
M1-G357, M1-N356, M1-Q355, M1-S354, M1-Y353, M1-I352, M1-V351,
M1-G350, M1-V349, M1-T348, M1-I347, M1-A346, M1-V345, M1-V344,
M1-I343, M1-A342, M1-P341, M1-V340, M1-G339, M1-W338, M1-G337,
M1-I336, M1-L335, M1-S334, M1-I333, M1-F332, M1-L331, M1-I330,
M1-F329, M1-H328, M1-R327, M1-P326, M1-L325, M1-P324, M1-K323,
M1-M322, M1-T321, M1-R320, M1-I319, M1-L318, M1-L317, M1-Y316,
M1-Y315, M1-L314, M1-Q313, M1-A312, M1-A311, M1-S310, M1-L309,
M1-A308, M1N307, M1-W306, M1-T305, M1-F304, M1-T303, M1-V302,
M1-L301, M1-L300, M1-F299, M1-Y298, M1-H297, M1-L296, M1-L295,
M1-A294, M1-A293, M1-I292, M1-A291, M1-T290, M1-C289, M1-M288,
M1-P287, M1-N286, M1-P285, M1-I284, M1-N283, M1-I282, M1-T281,
M1-D280, M1-T279, M1-R278, M1-P277, M1-I276, M1-D275, M1-N274,
M1-M273, M1-D272, M1-F271, M1-D270, M1-I269, M1-N268, M1-N267,
M1-I266, M1-D265, M1-G264, M1-D263, M1-S262, M1-T261, M1-Q260,
M1-L259, M1-N258, M1-K257, M1-N256, M1-S255, M1-N254, M1-E253,
M1-I252, M1-G251, M1-F250, M1-V249, M1-F248, M1-L247, M1-L246,
M1-N245, M1-F244, M1-I243, M1-L242, M1-M241, M1-S240, M1-I239,
M1-C238, M1-L237, M1-N236, M1-V235, M1-L234, M1-V233, M1-W232,
M1-T231, M1-V230, M1-S229, M1-T228, M1-K227, M1-R226, M1-V225,
M1-K224, M1-R223, M1-T222, M1-V221, M1-I220, M1-Q219, M1-F218,
M1-I217, M1-V216, M1-T215, M1-L214, M1-A213, M1-L212, M1-G211,
M1-T210, M1-V209, M1-S208, M1-L207, M1-A206, M1-C205, M1-G204,
M1-V203, M1-N202, M1-S201, M1-L200, M1-I199, M1-D198, M1-L197,
M1-S196, M1-K195, M1-P194, M1-Y193, M1-Q192, M1-Y191, M1-D190,
M1-K189, M1-K188, M1-F187, M1-T186, M1-M185, M1-L184, M1-V183,
M1-A182, M1-F181, M1-N180, M1-T179, M1-T178, M1-H177, M1-N176,
M1-C175, M1-R174, M1-C173, M1-R172, M1-L17l, M1-F170, M1-G169,
M1-D168, M1-T167, M1-G166, M1-K165, M1-D164, M1-K163, M1-Q162,
M1-C161, M1-G160, M1-Y159, M1-T158, M1-D157, M1-W156, M1-D155,
M1-K154, M1-A153, M1-S152, M1-L151, M1-N150, M1-W149, M1-Y148,
M1-V147, M1-C146, M1-A145, M1-Y144, M1-S143, M1-Y142, M1-L141,
M1-Q140, M1-F139, M1-E138, M1-K137, M1-Q136, M1-N135, M1-Y134,
M1-K133, M1-P132, M1-S131, M1-F130, M1-V129, M1-M128, M1-D127,
M1-V126, M1-S125, M1-A124, M1-S123, M1-Q122, M1-D121, M1-Q120,
M1-E119, M1-N118, M1-E117, M1-D116, M1-T115, M1-K114, M1-S133,
M1-S112, M-1-I111, M1-I110, M1-K109, M1-Q108, M1-S107, M1-F106,
M1-D105, M1-S104, M1-K103, M1-A102, M1-T101, M1-F100, M1-T99,
M1-K98, M1-S97, M1-Q96, M1-F95, M1-L94, M1-K93, M1-D92, M1-N91,
M1-Q90, M1-Y89, M1-V88, M1-V87, M1-F86, M1-G85, M1-C84, M1-T83,
M1-K82, M1-T81, M1-Y80, M1-N79, M1-K78, M1-T77, M1-M76, M1-N75,
M1-L74, M1-L73, M1-V72, M1-Q71, M1-L70, M1-E69, M1-T68, M1-Q67,
M1-A66, M1-D65, M1-P64, M1-N63, M1-L62, M1-G61, M1-D60, M1-V59,
M1-N58, M1-T57, M1-H56, M1-I55, M1-F54, M1-T53, M1-S52, M1-S51,
M1-S50, M1-V49, M1-L48, M1-S47, M1-S46, M1-S45, M1-A44, M1-G43,
M1-K42, M1-Q41, M1-V40, M1-S39, M1-F38, M1-R37, M1-V36, M1-N35,
M1-S34, M1-P33, M1-G32, M1-V31, M1-A30, M1-N29, M1-E28, M1-S27,
M1-S26, M1-F25, M1-N24, M1-A23, M1-S22, M1-Q21, M1-I20, M1-A19,
M1-I18, M1-N17, M1-P16, M1-E15, M1-V14, M1-V13, M1-S12, M1-Q11,
M1-N10, M1-G9, M1-L8, and/or M1-S7 of SEQ ID NO:2. Polynucleotide
sequences encoding these polypeptides are also included in SEQ ID
NO:1. The present invention also encompasses the use of these
C-terminal HGPRBMY6 deletion polypeptides as immunogenic and/or
antigenic epitopes as described elsewhere herein.
[0357] Alternatively, preferred polypeptides of the present
invention may comprise polypeptide sequences corresponding to, for
example, internal regions of the HGPRBMY6 polypeptide (e.g., any
combination of both N- and C-terminal HGPRBMY6 polypeptide
deletions) of SEQ ID NO:2. For example, internal regions could be
defined by the equation: amino acid NX to amino acid CX, wherein NX
refers to any N-terminal deletion polypeptide amino acid of
HGPRBMY6 (SEQ ID NO:2), and where CX refers to any C-terminal
deletion polypeptide amino acid of HGPRBMY6 (SEQ ID NO:2).
Polynucleotides encoding these polypeptides are also provided. The
present invention also encompasses the use of these polypeptides as
an immunogenic and/or antigenic epitope as described elsewhere
herein.
Example 9
Method of Enhancing the Biological Activity or Functional
Characteristics Through Molecular Evolution
[0358] Although many of the most biologically active proteins known
are highly effective for their specified function in an organism,
they often possess characteristics that make them undesirable for
transgenic, therapeutic, pharmaceutical, and/or industrial
applications. Among these traits, a short physiological half-life
is the most prominent problem, and is present either at the level
of the protein, or the level of the proteins mRNA. The ability to
extend the half-life, for example, would be particularly important
for a proteins use in gene therapy, transgenic animal production,
the bioprocess production and purification of the protein, and use
of the protein as a chemical modulator among others. Therefore,
there is a need to identify novel variants of isolated proteins
possessing characteristics which enhance their application as a
therapeutic for treating diseases of animal origin, in addition to
the proteins applicability to common industrial and pharmaceutical
applications.
[0359] Thus, one aspect of the present invention relates to the
ability to enhance specific characteristics of invention through
directed molecular evolution. Such an enhancement may, in a
non-limiting example, benefit the inventions utility as an
essential component in a kit, the inventions physical attributes
such as its solubility, structure, or codon optimization, the
inventions specific biological activity, including any associated
enzymatic activity, the proteins enzyme kinetics, the proteins Ki,
Kcat, Km, Vmax, Kd, protein-protein activity, protein-DNA binding
activity, antagonist/inhibitory activity (including direct or
indirect interaction), agonist activity (including direct or
indirect interaction), the proteins antigenicity (e.g., where it
would be desirable to either increase or decrease the antigenic
potential of the protein), the immunogenicity of the protein, the
ability of the protein to form dimers, trimers, or multimers with
either itself or other proteins, the antigenic efficacy of the
invention, including its subsequent use a preventative treatment
for disease or disease states, or as an effector for targeting
diseased genes. Moreover, the ability to enhance specific
characteristics of a protein may also be applicable to changing the
characterized activity of an enzyme to an activity completely
unrelated to its initially characterized activity. Other desirable
enhancements of the invention would be specific to each individual
protein, and would thus be well known in the art and contemplated
by the present invention.
[0360] For example, an engineered G-protein coupled receptor may be
constitutively active upon binding of its cognate ligand.
Alternatively, an engineered G-protein coupled receptor may be
constitutively active in the absence of ligand binding. In yet
another example, an engineered GPCR may be capable of being
activated with less than all of the regulatory factors and/or
conditions typically required for GPCR activation (e.g., ligand
binding, phosphorylation, conformational changes, etc.). Such GPCRs
would be useful in screens to identify GPCR modulators, among other
uses described herein.
[0361] Directed evolution is comprised of several steps. The first
step is to establish a library of variants for the gene or protein
of interest. The most important step is to then select for those
variants that entail the activity you wish to identify. The design
of the screen is essential since your screen should be selective
enough to eliminate non-useful variants, but not so stringent as to
eliminate all variants. The last step is then to repeat the above
steps using the best variant from the previous screen. Each
successive cycle, can then be tailored as necessary, such as
increasing the stringency of the screen, for example.
[0362] Over the years, there have been a number of methods
developed to introduce mutations into macromolecules. Some of these
methods include, random mutagenesis, "error-prone" PCR, chemical
mutagenesis, site-directed mutagenesis, and other methods well
known in the art (for a comprehensive listing of current
mutagenesis methods, see Maniatis, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982)).
Typically, such methods have been used, for example, as tools for
identifying the core functional region(s) of a protein or the
function of specific domains of a protein (if a multi-domain
protein). However, such methods have more recently been applied to
the identification of macromolecule variants with specific or
enhanced characteristics.
[0363] Random mutagenesis has been the most widely recognized
method to date. Typically, this has been carried out either through
the use of "error-prone" PCR (as described in Moore, J., et al,
Nature Biotechnology 14:458, (1996), or through the application of
randomized synthetic oligonucleotides corresponding to specific
regions of interest (as descibed by Derbyshire, K. M. et al, Gene,
46:145-152, (1986), and Hill, D E, et al, Methods Enzymol.,
55:559-568, (1987). Both approaches have limits to the level of
mutagenesis that can be obtained. However, either approach enables
the investigator to effectively control the rate of mutagenesis.
This is particularly important considering the fact that mutations
beneficial to the activity of the enzyme are fairly rare. In fact,
using too high a level of mutagenesis may counter or inhibit the
desired benefit of a useful mutation.
[0364] While both of the aforementioned methods are effective for
creating randomized pools of macromolecule variants, a third
method, termed "DNA Shuffling", or "sexual PCR" (W P C, Stemmer,
PNAS, 91:10747, (1994)) has recently been elucidated. DNA shuffling
has also been referred to as "directed molecular evolution",
"exon-shuffling", "directed enzyme evolution", "in vitro
evolution", and "artificial evolution". Such reference terms are
known in the art and are encompassed by the invention. This new,
preferred, method apparently overcomes the limitations of the
previous methods in that it not only propagates positive traits,
but simultaneously eliminates negative traits in the resulting
progeny.
[0365] DNA shuffling accomplishes this task by combining the
principal of in vitro recombination, along with the method of
"error-prone" PCR. In effect, you begin with a randomly digested
pool of small fragments of your gene, created by Dnase I digestion,
and then introduce said random fragments into an "error-prone" PCR
assembly reaction. During the PCR reaction, the randomly sized DNA
fragments not only hybridize to their cognate strand, but also may
hybridize to other DNA fragments corresponding to different regions
of the polynucleotide of interest--regions not typically accessible
via hybridization of the entire polynucleotide. Moreover, since the
PCR assembly reaction utilizes "error-prone" PCR reaction
conditions, random mutations are introduced during the DNA
synthesis step of the PCR reaction for all of the
fragments--further diversifying the potential hybridation sites
during the annealing step of the reaction.
[0366] A variety of reaction conditions could be utilized to
carry-out the DNA shuffling reaction. However, specific reaction
conditions for DNA shuffling are provided, for example, in PNAS,
91:10747, (1994). Briefly, the DNA substrate to be subjected to the
DNA shuffling reaction is prepared. Preparation may be in the form
of simply purifying the DNA from contaminating cellular material,
chemicals, buffers, oligonucleotide primers, deoxynucleotides,
RNAs, etc., and may entail the use of DNA purification kits as
those provided by Qiagen, Inc., or by the Promega, Corp., for
example.
[0367] Once the DNA substrate has been purified, it would be
subjected to Dnase I digestion. About 2-4 ug of the DNA
substrate(s) would be digested with 0.0015 units of Dnase I (Sigma)
per ul in 100 ul of 50 mM Tris-HCL, pH 7.4/1 mM MgCl2 for 10-20
min. at room temperature. The resulting fragments of 10-50 bp could
then be purified by running them through a 2% low-melting point
agarose gel by electrophoresis onto DE81 ion-exchange paper
(Whatman) or could be purified using Microcon concentrators
(Amicon) of the appropriate molecular weight cuttoff, or could use
oligonucleotide purification columns (Qiagen), in addition to other
methods known in the art. If using DE81 ion-exchange paper, the
10-50 bp fragments could be eluted from said paper using 1M NaCL,
followed by ethanol precipitation.
[0368] The resulting purified fragments would then be subjected to
a PCR assembly reaction by re-suspension in a PCR mixture
containing: 2 mM of each dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 mM Tris.
HCL, pH 9.0, and 0.1% Triton X-100, at a final fragment
concentration of 10-30 ng/ul. No primers are added at this point.
Taq DNA polymerase (Promega) would be used at 2.5 units per 100 ul
of reaction mixture. A PCR program of 94 C. for 60 s; 94 C. for 30
s, 50-55 C. for 30 s, and 72 C. for 30 s using 30-45 cycles,
followed by 72 C. for 5 min using an MJ Research (Cambridge, Mass.)
PTC-150 thermocycler. After the assembly reaction is completed, a
1:40 dilution of the resulting primerless product would then be
introduced into a PCR mixture (using the same buffer mixture used
for the assembly reaction) containing 0.8 um of each primer and
subjecting this mixture to 15 cycles of PCR (using 94 C. for 30 s,
50 C. for 30 s, and 72 C. for 30 s). The referred primers would be
primers corresponding to the nucleic acid sequences of the
polynucleotide(s) utilized in the shuffling reaction. Said primers
could consist of modified nucleic acid base pairs using methods
known in the art and referred to else where herein, or could
contain additional sequences (i.e., for adding restriction sites,
mutating specific base-pairs, etc.).
[0369] The resulting shuffled, assembled, and amplified product can
be purified using methods well known in the art (e.g., Qiagen PCR
purification kits) and then subsequently cloned using appropriate
restriction enzymes.
[0370] Although a number of variations of DNA shuffling have been
published to date, such variations would be obvious to the skilled
artisan and are encompassed by the invention. The DNA shuffling
method can also be tailered to the desired level of mutagenesis
using the methods described by Zhao, et al. Nucl. Acid Res., 25(6):
1307-1308, (1997).
[0371] As described above, once the randomized pool has been
created, it can then be subjected to a specific screen to identify
the variant possessing the desired characteristic(s). Once the
variant has been identified, DNA corresponding to the variant could
then be used as the DNA substrate for initiating another round of
DNA shuffling. This cycle of shuffling, selecting the optimized
variant of interest, and then re-shuffling, can be repeated until
the ultimate variant is obtained. Examples of model screens applied
to identify variants created using DNA shuffling technology may be
found in the following publications: J. C., Moore, et al., J. Mol.
Biol., 272:336-347, (1997), F. R., Cross, et al., Mol. Cell. Biol.,
18:2923-2931, (1998), and A. Crameri., et al., Nat. Biotech.,
15:436-438, (1997).
[0372] DNA shuffling has several advantages. First, it makes use of
beneficial mutations. When combined with screening, DNA shuffling
allows the discovery of the best mutational combinations and does
not assume that the best combination contains all the mutations in
a population. Secondly, recombination occurs simultaneously with
point mutagenesis. An effect of forcing DNA polymerase to
synthesize full-length genes from the small fragment DNA pool is a
background mutagenesis rate. In combination with a stringent
selection method, enzymatic activity has been evolved up to 16000
fold increase over the wild-type form of the enzyme. In essence,
the background mutagenesis yielded the genetic variability on which
recombination acted to enhance the activity.
[0373] A third feature of recombination is that it can be used to
remove deleterious mutations. As discussed above, during the
process of the randomization, for every one beneficial mutation,
there may be at least one or more neutral or inhibitory mutations.
Such mutations can be removed by including in the assembly reaction
an excess of the wild-type random-size fragments, in addition to
the random-size fragments of the selected mutant from the previous
selection. During the next selection, some of the most active
variants of the polynucleotide/polypeptide/enzyme- , should have
lost the inhibitory mutations.
[0374] Finally, recombination enables parallel processing. This
represents a significant advantage since there are likely multiple
characteristics that would make a protein more desirable (e.g.
solubility, activity, etc.). Since it is increasingly difficult to
screen for more than one desirable trait at a time, other methods
of molecular evolution tend to be inhibitory. However, using
recombination, it would be possible to combine the randomized
fragments of the best representative variants for the various
traits, and then select for multiple properties at once.
[0375] DNA shuffling can also be applied to the polynucleotides and
polypeptides of the present invention to decrease their
immunogenicity in a specified host. For example, a particular
varient of the present invention may be created and isolated using
DNA shuffling technology. Such a variant may have all of the
desired characteristics, though may be highly immunogenic in a host
due to its novel intrinsic structure. Specifically, the desired
characteristic may cause the polypeptide to have a non-native
strucuture which could no longer be recognized as a "self"
molecule, but rather as a "foreign", and thus activate a host
immune response directed against the novel variant. Such a
limitation can be overcome, for example, by including a copy of the
gene sequence for a xenobiotic ortholog of the native protein in
with the gene sequence of the novel variant gene in one or more
cycles of DNA shuffling. The molar ratio of the ortholog and novel
variant DNAs could be varied accordingly. Ideally, the resulting
hybrid variant identified would contain at least some of the coding
sequence which enabled the xenobiotic protein to evade the host
immune system, and additionally, the coding sequence of the
original novel varient that provided the desired
characteristics.
[0376] Likewise, the invention encompasses the application of DNA
shuffling technology to the evolution of polynucletotides and
polypeptides of the invention, wherein one or more cycles of DNA
shuffling include, in addition to the gene template DNA,
oligonucleotides coding for known allelic sequences, optimized
codon sequences, known variant sequences, known polynucleotide
polymorphism sequences, known ortholog sequences, known homolog
sequences, additional homologous sequences, additional
non-homologous sequences, sequences from another species, and any
number and combination of the above.
[0377] In addition to the described methods above, there are a
number of related methods that may also be applicable, or desirable
in certain cases. Representative among these are the methods
discussed in PCT applications WO 98/31700, and WO 98/32845, which
are hereby incorporated by reference. Furthermore, related methods
can also be applied to the polynucleotide sequences of the present
invention in order to evolve invention for creating ideal variants
for use in gene therapy, protein engineering, evolution of whole
cells containing the variant, or in the evolution of entire enzyme
pathways containing polynucleotides of the invention as described
in PCT applications WO 98/13485, WO 98/13487, WO 98/27230, WO
98/31837, and Crameri, A., et al., Nat. Biotech., 15:436-438,
(1997), respectively.
[0378] Additional methods of applying "DNA Shuffling" technology to
the polynucleotides and polypeptides of the present invention,
including their proposed applications, may be found in U.S. Pat.
No. 5,605,793; PCT Application No. WO 95/22625; PCT Application No.
WO 97/20078; PCT Application No. WO 97/35966; and PCT Application
No. WO 98/42832; PCT Application No. WO 00/09727 specifically
provides methods for applying DNA shuffling to the identification
of herbicide selective crops which could be applied to the
polynucleotides and polypeptides of the present invention;
additionally, PCT Application No. WO 00/12680 provides methods and
compositions for generating, modifying, adapting, and optimizing
polynucleotide sequences that confer detectable phenotypic
properties on plant species; each of the above are hereby
incorporated in their entirety herein for all purposes.
[0379] The contents of all patents, patent applications, published
PCT applications and articles, books, references, reference manuals
and abstracts cited herein are hereby incorporated by reference in
their entirety to more fully describe the state of the art to which
the invention pertains.
[0380] As various changes can be made in the above-described
subject matter without departing from the scope and spirit of the
present invention, it is intended that all subject matter contained
in the above description, or defined in the appended claims, be
interpreted as descriptive and illustrative of the present
invention. Many modifications and variations of the present
invention are possible in light of the above teachings.
[0381] References
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for the study of G Protein Coupled Receptor signalling in mammalian
cells". In Milligan G. (ed.): Signal Transduction: A practical
approach. Oxford: Oxford University Press, 1999: 171-221.
[0383] 2. Alam, J., Cook, J. L.: "Reporter Genes: Application to
the study of mammalian gene transcription". Anal. Biochem. 1990;
188: 245-254.
[0384] 3. Selbie, L. A. and Hill, S. J.: "G protein-coupled
receptor cross-talk: The fine-tuning of multiple receptor-signaling
pathways". TiPs. 1998; 19: 87-93.
[0385] 4. Boss, V., Talpade, D. J., and Murphy, T. J.: "Induction
of NFAT mediated transcription by Gq-coupled Receptors in lympoid
and non-lymphoid cells". JBC. 1996; 271: 10429-10432.
[0386] 5. George, S. E., Bungay, B. J., and Naylor, L. H.:
"Functional coupling of endogenous serotonin (5-HT1B) and
calcitonin (C1a) receptors in CHO cells to a cyclic AMP-responsive
luciferase reporter gene". J. Neurochem. 1997; 69: 1278-1285.
[0387] 6. Suto, C M, Igna D M: "Selection of an optimal reporter
for cell-based high throughput screening assays". J. Biomol.
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[0388] 7. Zlokarnik, G., Negulescu, P. A., Knapp, T. E., More, L.,
Burres, N., Feng, L., Whitney, M., Roemer, K., and Tsien, R. Y.
"Quantitation of transcription and clonal selection of single
living cells with a B-Lactamase Reporter". Science. 1998; 279:
84-88.
[0389] 8. S. Fiering et. al., Genes Dev. 4, 1823 (1990).
[0390] 9. J. Karttunen and N. Shastri, PNAS 88, 3972 (1991).
[0391] 10. Hawes, B. E., Luttrell. L. M., van Biesen, T., and
Lefkowitz, R. J. (1996) JBC 271, 12133-12136.
[0392] 11. Gilman, A. G. (1987) Annul. Rev. Biochem. 56,
615-649.
[0393] 12. Maniatis et al., Cold Spring Harbor Press, 1989.
[0394] 13. Salcedo, R., Ponce, M. L., Young, H. A., Wasserman, K.,
Ward, J. M., Kleinman, H. K., Oppenheim, J. J., Murphy, W. J.
"Human endothelial cells express CCF2 and respond to MCP-1: direct
role of MCP-1 in angiogenesis and tumor progression". Blood. 2000;
96 (1): 34-40.
[0395] 14. Sica, A., Saccani, A., Bottazzi, B., Bernasconi, S.,
Allavena, P., Gaetano, B., LaRossa, G., Scotton, C., Balkwill F.,
Mantovani, A. "Defective expression of the monocyte chemotactic
protein 1 receptor CCF2 in macrophages associated with human
ovarian carcinoma". J. Immunology. 2000; 164: 733-8.
[0396] 15. Kypson, A., Hendrickson, S., Akhter, S., Wilson, K.,
McDonald, P., Lilly, R., Dolber, P., Glower, D., Lefkowitz, R.,
Koch, W. "Adenovirus-mediated gene transfer of the B2 AR to donor
hearts enhances cardiac function". Gene Therapy. 1999; 6:
1298-304.
[0397] 16. Dorn, G. W., Tepe, N. M., Lorenz, J. N., Kock, W. J.,
Ligget, S. B. "Low and high level transgenic expression of B2AR
differentially affect cardiac hypertrophy and function in Galpha
q-overexpressing mice". PNAS. 1999; 96: 6400-5.
[0398] 17. J. Wess. "G protein coupled receptor: molecular
mechanisms involved in receptor activation and selectivity of
G-protein recognition". FASEB. 1997; 11:346-354.
[0399] 18. Whitney, M, Rockenstein, E, Cantin, G., Knapp, T.,
Zlokarnik, G., Sanders, P., Durick, K., Craig, F. F., and
Negulescu, P. A. "A genome-wide functional assay of signal
transduction in living mammalian cells". Nature Biotech. 1998; 16:
1329-1333.
[0400] 19. BD Biosciences: FACS Vantage SE Training Manual. Part
Number 11-11020-00 Rev. A. August 1999.
[0401] 20. Chen, G., Jaywickreme, C., Way, J., Armour S., Queen K.,
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Receptor systems for drug discovery". J. Pharmacol. Toxicol.
Methods 1999; 42: 199-206.
[0402] 21. Blahos, J., Fischer, T., Brabet, I., Stauffer, D.,
Rovelli, G., Bockaert, J., and Pin, J.-P. "A novel Site on the G
alpha-protein that Rocognized Heptahelical Receptors". J.Biol.
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Biol. Chem. 1995; 270, No. 25, 15175-80.
Sequence CWU 1
1
81 1 1683 DNA Homo sapiens 1 atggagactt attccttgtc tttgggtaat
caatcagtgg tggaacctaa catagcaata 60 cagtcagcaa atttctcttc
agaaaatgcg gtggggcctt caaatgttcg cttctctgtg 120 cagaaaggag
ctagcagttc tctagtttct agttcaacat ttatacatac aaatgtggat 180
ggccttaacc cagatgcaca gactgagctt caggtcttgc ttaatatgac gaaaaattac
240 accaagacat gcggctttgt agtttatcaa aatgacaagc ttttccaatc
aaaaactttt 300 acagctaaat cggattttag tcaaaaaatt atctcaagca
aaactgatga aaatgagcaa 360 gatcagagtg cttctgttga catggtcttt
agtccaaagt acaaccaaaa agaatttcaa 420 ctctattcct atgcctgtgt
ctattggaat ttgtcagcga aggactggga cacatatggc 480 tgtcaaaaag
acaagggcac tgatggattc ctgcgctgcc gctgcaacca tactactaat 540
tttgctgtat taatgacttt caaaaaggat tatcaatatc ccaaatcact tgacatatta
600 tccaacgttg gatgtgcact gtctgttact ggtctggctc tcacagttat
atttcagatt 660 gtcaccagga aagtcagaaa aacctcagta acctgggttt
tggtcaatct gtgcatatca 720 atgttgattt tcaacctcct ctttgtgttt
ggaattgaaa actccaataa gaacttgcag 780 acaagtgatg gtgacatcaa
taatattgac tttgacaata atgacatacc caggacagac 840 accattaaca
tcccgaatcc catgtgcact gcgattgccg ccttactgca ctattttctg 900
ttagtgacat ttacctggaa cgcactcagc gctgcacagc tctattacct tctaataagg
960 accatgaagc ctcttcctcg gcatttcatt cttttcatct cattaattgg
atggggagtc 1020 ccagctatag tagtggctat aacagtggga gttatttatt
ctcagaatgg aaataatcca 1080 cagtgggaat tagactaccg gcaagagaaa
atctgctggc tggcaattcc agaacccaat 1140 ggtgttataa aaagtccgct
gttgtggtca ttcatcgtac ctgtaaccat tatcctcatc 1200 agcaatgttg
ttatgtttat tacaatctcg atcaaagtgc tgtggaagaa taaccagaac 1260
ctgacaagca caaaaaaagt ttcatccatg aagaagattg ttagcacatt atctgttgca
1320 gttgtttttg gaattacctg gattctagca tacctgatgc tagttaatga
tgatagcatc 1380 aggatcgtct tcagctacat attctgcctt ttcaacacta
cacagggatt gcaaattttt 1440 atcctgtaca ctgttagaac aaaagtcttc
cagagtgaag cttccaaagt gttgatgttg 1500 ctatcgtcta ttgggagaag
gaagtcattg ccttcagtga cgcggccgag gctgcgtgta 1560 aagatgtata
atttcctcag gtcattgcca accttacatg aacgctttag gctactggaa 1620
acctctccga gtactgagga aatcacactc tctgaaagtg acaatgcaaa ggaaagcatc
1680 tag 1683 2 560 PRT Homo sapiens 2 Met Glu Thr Tyr Ser Leu Ser
Leu Gly Asn Gln Ser Val Val Glu Pro 1 5 10 15 Asn Ile Ala Ile Gln
Ser Ala Asn Phe Ser Ser Glu Asn Ala Val Gly 20 25 30 Pro Ser Asn
Val Arg Phe Ser Val Gln Lys Gly Ala Ser Ser Ser Leu 35 40 45 Val
Ser Ser Ser Thr Phe Ile His Thr Asn Val Asp Gly Leu Asn Pro 50 55
60 Asp Ala Gln Thr Glu Leu Gln Val Leu Leu Asn Met Thr Lys Asn Tyr
65 70 75 80 Thr Lys Thr Cys Gly Phe Val Val Tyr Gln Asn Asp Lys Leu
Phe Gln 85 90 95 Ser Lys Thr Phe Thr Ala Lys Ser Asp Phe Ser Gln
Lys Ile Ile Ser 100 105 110 Ser Lys Thr Asp Glu Asn Glu Gln Asp Gln
Ser Ala Ser Val Asp Met 115 120 125 Val Phe Ser Pro Lys Tyr Asn Gln
Lys Glu Phe Gln Leu Tyr Ser Tyr 130 135 140 Ala Cys Val Tyr Trp Asn
Leu Ser Ala Lys Asp Trp Asp Thr Tyr Gly 145 150 155 160 Cys Gln Lys
Asp Lys Gly Thr Asp Gly Phe Leu Arg Cys Arg Cys Asn 165 170 175 His
Thr Thr Asn Phe Ala Val Leu Met Thr Phe Lys Lys Asp Tyr Gln 180 185
190 Tyr Pro Lys Ser Leu Asp Ile Leu Ser Asn Val Gly Cys Ala Leu Ser
195 200 205 Val Thr Gly Leu Ala Leu Thr Val Ile Phe Gln Ile Val Thr
Arg Lys 210 215 220 Val Arg Lys Thr Ser Val Thr Trp Val Leu Val Asn
Leu Cys Ile Ser 225 230 235 240 Met Leu Ile Phe Asn Leu Leu Phe Val
Phe Gly Ile Glu Asn Ser Asn 245 250 255 Lys Asn Leu Gln Thr Ser Asp
Gly Asp Ile Asn Asn Ile Asp Phe Asp 260 265 270 Asn Asn Asp Ile Pro
Arg Thr Asp Thr Ile Asn Ile Pro Asn Pro Met 275 280 285 Cys Thr Ala
Ile Ala Ala Leu Leu His Tyr Phe Leu Leu Val Thr Phe 290 295 300 Thr
Trp Asn Ala Leu Ser Ala Ala Gln Leu Tyr Tyr Leu Leu Ile Arg 305 310
315 320 Thr Met Lys Pro Leu Pro Arg His Phe Ile Leu Phe Ile Ser Leu
Ile 325 330 335 Gly Trp Gly Val Pro Ala Ile Val Val Ala Ile Thr Val
Gly Val Ile 340 345 350 Tyr Ser Gln Asn Gly Asn Asn Pro Gln Trp Glu
Leu Asp Tyr Arg Gln 355 360 365 Glu Lys Ile Cys Trp Leu Ala Ile Pro
Glu Pro Asn Gly Val Ile Lys 370 375 380 Ser Pro Leu Leu Trp Ser Phe
Ile Val Pro Val Thr Ile Ile Leu Ile 385 390 395 400 Ser Asn Val Val
Met Phe Ile Thr Ile Ser Ile Lys Val Leu Trp Lys 405 410 415 Asn Asn
Gln Asn Leu Thr Ser Thr Lys Lys Val Ser Ser Met Lys Lys 420 425 430
Ile Val Ser Thr Leu Ser Val Ala Val Val Phe Gly Ile Thr Trp Ile 435
440 445 Leu Ala Tyr Leu Met Leu Val Asn Asp Asp Ser Ile Arg Ile Val
Phe 450 455 460 Ser Tyr Ile Phe Cys Leu Phe Asn Thr Thr Gln Gly Leu
Gln Ile Phe 465 470 475 480 Ile Leu Tyr Thr Val Arg Thr Lys Val Phe
Gln Ser Glu Ala Ser Lys 485 490 495 Val Leu Met Leu Leu Ser Ser Ile
Gly Arg Arg Lys Ser Leu Pro Ser 500 505 510 Val Thr Arg Pro Arg Leu
Arg Val Lys Met Tyr Asn Phe Leu Arg Ser 515 520 525 Leu Pro Thr Leu
His Glu Arg Phe Arg Leu Leu Glu Thr Ser Pro Ser 530 535 540 Thr Glu
Glu Ile Thr Leu Ser Glu Ser Asp Asn Ala Lys Glu Ser Ile 545 550 555
560 3 2212 DNA Homo sapiens 3 ccacgctttc cctccctgac cacaggtgat
ccgcctgcct cagcctcccg aagtgcaggg 60 attacaggcg tagtaagtaa
gccaccacac ctggccgcca ctcttatttt taaaagttga 120 catcagtttg
tgaaaaagga ctgttgtttc atcaaatttc agcaaatgat gatcaatagc 180
acattaaaaa tggcttcatc tttgtggaag ttttgactgg atatagatcc ctgacatttg
240 agaccaaagg aaagcctctt gatggtgtaa ctggaccaga atgaagagaa
agaaactatt 300 atcaaagacc cttggaaaca ggaaactcca aacctgatgc
gggtctcagg gcagtatcta 360 tgagcaggtg aaatagaaag tacatctaac
tagatgtttt ttcatgcaga ttaaattatt 420 ttgaccaaag ttgtacccaa
atgcacatgc atggaagagc taacactagg ggacaagcaa 480 gggggaggaa
gaggaaacca acctttatgt acagcctttc atgtgcctgg catgttgcat 540
atgttatcac atttaatcct tataaaactt ctgtgagttg aatgttattc ccatattata
600 aataattata gccaataaca cttactaatt gttgagcacc tactgcatgc
caaatattgt 660 gccaaatatt aatgtattta ttagtttatc atatttaatt
tttataacac cataaatagg 720 tattaatgta cacattttat agatgaggaa
aatgtggttc tgagaggtga agcattttgc 780 ctagtgatca cagctaaaaa
gtgatagagc tgttctttat tttaaagttc acattgtact 840 accctggctc
cctaatcaca gatgggcagg gtaggggttg ggtggggaca gaagttggag 900
agtggatgtg gctgccaacc acacaagttg tgccaaccca cagattgagg aaagatgcta
960 aatttggaat ctggcaaacc agtgtttggt tcttagctct gccacttcta
agctgtgtga 1020 aacttggttg aggtccctaa cttctcctga gggtgaacaa
ctcacaaagt tgttttgctt 1080 attaaatgtg ataacacctg taaacatcta
acagagtgcc tagcacatag cagggatcta 1140 gcaattgaat tagggttatt
tgtttctgtc tactgattgg gtattgtttc tgacacttac 1200 ccaagtgtga
atagcctata acactggtat aatttgtgaa atgatgctgc catctagtga 1260
aaaccaagac acacacacac acacacacac acacacacat acacacacac gtgcgcgcgc
1320 atggacaccc agcttcacca atgacaatat ggattggcat gttttagcct
cacaacacag 1380 agccctgggg ctaactggca cctagagagg tcatctcggc
cagtgccttc caaactacca 1440 gtgctgaaaa gccagttcaa aaaattttga
acccattgca caccaatatt tttgtgaaat 1500 accataaaaa taaattactg
gaaaaatgaa ataaaaaata tgtataaaat acaaaccaaa 1560 attttagaac
tgttagattc aacagcaaaa aattgctgta tacatctctg accaattgct 1620
ttcagtttct gtgcttatct ctctacgacc tttgtaacac acagtgaacc agcgctggcc
1680 catggataca ctctagtagc cccaatctag ctaaggcagc cccttatagt
taatcaatcc 1740 tgtcaaacag gaaaggctgg caaaaccact ggtctgcatg
tactttgtcc tttacacaag 1800 gaaggatgca aacgtggaaa actgagtgga
catggtgttc aggagattga ggctcagcta 1860 aattccagct tatttacctg
cagttgctta caaagtgttt ggacataatt gtgtaaagct 1920 agggtttttt
ttctggtttt taaaacaggt aaaggatgtc acagcaccac ttaataacat 1980
ttcttctgaa gtccagattt taacatctga tgccaataaa ttaactgctg agaacatcac
2040 tagtgctacg cgagtggttg gacagatatt caacacttcc agaaatgctt
cacctgaggc 2100 aaagaaagtt gccatagtaa cagtgagtca actcctagat
gccagtgaag atgcttttca 2160 aagagttgct gctactgcta atgatgatgc
ccttacaacg cttattgagc aa 2212 4 449 DNA Homo sapiens 4 acagtaaaac
ttacctgttg tggtcttttt aatcacctcg tttgagtttt atctgtttct 60
ctcctttatt tcccagtcct ctcagaaagt cttcctcaat gtattttgct caggattaag
120 aattagataa aacctgttgt ttattattat tcggcataat ggacttggta
gtttttctat 180 ttttcaatag atttgtactt gaataaggtg aagaatttca
cacaacatac aagagtacca 240 ttgttcctta tatcgttaaa tctttgtgac
acactttgac aaaaatgtag aacctataac 300 aaattctttt acaagttact
ataaaggaca caaagagaaa actttacctt ccagaacaaa 360 atgactcctg
atgaacagtg tgtggggatt tgcttgtatg tattaaactt ttgacctctg 420
aaaaaaaaaa aaaaaaaaaa aaaaaaaag 449 5 80 DNA Artificial Sequence
Synthetic Oligos 5 gctgtgcagc gctgagtgcg ttccaggtaa atgtcactaa
cagaaaatag tgcagtaagg 60 cggcaatcgc agtgcacatg 80 6 23 DNA
Artificial Sequence Synthetic Oligos 6 cagacaccat taacatcccg aat 23
7 22 DNA Artificial Sequence Synthetic Oligos 7 agaatgaaat
gccgaggaag ag 22 8 1230 PRT Rat 8 Met Cys Pro Pro Gln Leu Phe Ile
Leu Met Met Leu Leu Ala Pro Val 1 5 10 15 Val His Ala Phe Ser Arg
Ala Pro Ile Pro Met Ala Val Val Arg Arg 20 25 30 Glu Leu Ser Cys
Glu Ser Tyr Pro Ile Glu Leu Arg Cys Pro Gly Thr 35 40 45 Asp Val
Ile Met Ile Glu Ser Ala Asn Tyr Gly Arg Thr Asp Asp Lys 50 55 60
Ile Cys Asp Ser Asp Pro Ala Gln Met Glu Asn Ile Arg Cys Tyr Leu 65
70 75 80 Pro Asp Ala Tyr Lys Ile Met Ser Gln Arg Cys Asn Asn Arg
Thr Gln 85 90 95 Cys Ala Val Val Ala Gly Pro Asp Val Phe Pro Asp
Pro Cys Pro Gly 100 105 110 Thr Tyr Lys Tyr Leu Glu Val Gln Tyr Glu
Cys Val Pro Tyr Lys Val 115 120 125 Glu Gln Lys Val Phe Leu Cys Pro
Gly Leu Leu Lys Gly Val Tyr Gln 130 135 140 Ser Glu His Leu Phe Glu
Ser Asp His Gln Ser Gly Ala Trp Cys Lys 145 150 155 160 Asp Pro Leu
Gln Ala Ser Asp Lys Ile Tyr Tyr Met Pro Trp Thr Pro 165 170 175 Tyr
Arg Thr Asp Thr Leu Thr Glu Tyr Ser Ser Lys Asp Asp Phe Ile 180 185
190 Ala Gly Arg Pro Thr Thr Thr Tyr Lys Leu Pro His Arg Val Asp Gly
195 200 205 Thr Gly Phe Val Val Tyr Asp Gly Ala Leu Phe Phe Asn Lys
Glu Arg 210 215 220 Thr Arg Asn Ile Val Lys Phe Asp Leu Arg Thr Arg
Ile Lys Ser Gly 225 230 235 240 Glu Ala Ile Ile Ala Asn Ala Asn Tyr
His Asp Thr Ser Pro Tyr Arg 245 250 255 Trp Gly Gly Lys Ser Asp Ile
Asp Leu Ala Val Asp Glu Asn Gly Leu 260 265 270 Trp Val Ile Tyr Ala
Thr Glu Gln Asn Asn Gly Lys Ile Val Ile Ser 275 280 285 Gln Leu Asn
Pro Tyr Thr Leu Arg Ile Glu Gly Thr Trp Asp Thr Ala 290 295 300 Tyr
Asp Lys Arg Ser Ala Ser Asn Ala Phe Met Ile Cys Gly Ile Leu 305 310
315 320 Tyr Val Val Lys Ser Val Tyr Glu Asp Asp Asp Asn Glu Ala Thr
Gly 325 330 335 Asn Lys Ile Asp Tyr Ile Tyr Asn Thr Asp Gln Ser Lys
Asp Ser Leu 340 345 350 Val Asp Val Pro Phe Pro Asn Ser Tyr Gln Tyr
Ile Ala Ala Val Asp 355 360 365 Tyr Asn Pro Arg Asp Asn Leu Leu Tyr
Val Trp Asn Asn Tyr His Val 370 375 380 Val Lys Tyr Ser Leu Asp Phe
Gly Pro Leu Asp Ser Arg Ser Gly Pro 385 390 395 400 Val His His Gly
Gln Val Ser Tyr Ile Ser Pro Pro Ile His Leu Asp 405 410 415 Ser Asp
Leu Glu Arg Pro Pro Val Arg Gly Ile Ser Thr Thr Gly Pro 420 425 430
Leu Gly Met Gly Ser Thr Thr Thr Ser Thr Thr Leu Arg Thr Thr Thr 435
440 445 Trp Asn Leu Gly Arg Ser Thr Thr Pro Ser Leu Pro Gly Arg Arg
Asn 450 455 460 Arg Ser Thr Ser Thr Pro Ser Pro Ala Ile Glu Val Leu
Asp Val Thr 465 470 475 480 Thr His Leu Pro Ser Ala Ala Ser Gln Ile
Pro Ala Met Glu Glu Ser 485 490 495 Cys Glu Ala Val Glu Ala Arg Glu
Ile Met Trp Phe Lys Thr Arg Gln 500 505 510 Gly Gln Val Ala Lys Gln
Ser Cys Pro Ala Gly Thr Ile Gly Val Ser 515 520 525 Thr Tyr Leu Cys
Leu Ala Pro Asp Gly Ile Trp Asp Pro Gln Gly Pro 530 535 540 Asp Leu
Ser Asn Cys Ser Ser Pro Trp Val Asn His Ile Thr Gln Lys 545 550 555
560 Leu Lys Ser Gly Glu Thr Ala Ala Asn Ile Ala Arg Glu Leu Ala Glu
565 570 575 Gln Thr Arg Asn His Leu Asn Ala Gly Asp Ile Thr Tyr Ser
Val Arg 580 585 590 Ala Met Asp Gln Leu Val Gly Leu Leu Asp Val Gln
Leu Arg Asn Leu 595 600 605 Thr Pro Gly Gly Lys Asp Ser Ala Ala Arg
Ser Leu Asn Lys Leu Gln 610 615 620 Lys Arg Glu Arg Ser Cys Arg Ala
Tyr Val Gln Ala Met Val Glu Thr 625 630 635 640 Val Asn Asn Leu Leu
Gln Pro Gln Ala Leu Asn Ala Trp Arg Asp Leu 645 650 655 Thr Thr Ser
Asp Gln Leu Arg Ala Ala Thr Met Leu Leu Asp Thr Val 660 665 670 Glu
Glu Ser Ala Phe Val Leu Ala Asp Asn Leu Leu Lys Thr Asp Ile 675 680
685 Val Arg Glu Asn Thr Asp Asn Ile Gln Leu Glu Val Ala Arg Leu Ser
690 695 700 Thr Glu Gly Asn Leu Glu Asp Leu Lys Phe Pro Glu Asn Thr
Gly His 705 710 715 720 Gly Ser Thr Ile Gln Leu Ser Ala Asn Thr Leu
Lys Gln Asn Gly Arg 725 730 735 Asn Gly Glu Ile Arg Val Ala Phe Val
Leu Tyr Asn Asn Leu Gly Pro 740 745 750 Tyr Leu Ser Thr Glu Asn Ala
Ser Met Lys Leu Gly Thr Glu Ala Met 755 760 765 Ser Thr Asn His Ser
Val Ile Val Asn Ser Pro Val Ile Thr Ala Ala 770 775 780 Ile Asn Lys
Glu Phe Ser Asn Lys Val Tyr Leu Ala Asp Pro Val Val 785 790 795 800
Phe Thr Val Lys His Ile Lys Gln Ser Glu Glu Asn Phe Asn Pro Asn 805
810 815 Cys Ser Phe Trp Ser Tyr Ser Lys Arg Thr Met Thr Gly Tyr Trp
Ser 820 825 830 Thr Gln Gly Cys Arg Leu Leu Thr Thr Asn Lys Thr His
Thr Thr Cys 835 840 845 Ser Cys Asn His Leu Thr Asn Phe Ala Val Leu
Met Ala His Val Glu 850 855 860 Val Lys His Ser Asp Ala Val His Asp
Leu Leu Leu Asp Val Ile Thr 865 870 875 880 Trp Val Gly Ile Leu Leu
Ser Leu Val Cys Leu Leu Ile Cys Ile Phe 885 890 895 Thr Phe Cys Phe
Phe Arg Gly Leu Gln Ser Asp Arg Asn Thr Ile His 900 905 910 Lys Asn
Leu Cys Ile Ser Leu Phe Val Ala Glu Leu Leu Phe Leu Ile 915 920 925
Gly Ile Asn Arg Thr Asp Gln Pro Ile Ala Cys Ala Val Phe Ala Ala 930
935 940 Leu Leu His Phe Phe Phe Leu Ala Ala Phe Thr Trp Met Phe Leu
Glu 945 950 955 960 Gly Val Gln Leu Tyr Ile Met Leu Val Glu Val Phe
Glu Ser Glu His 965 970 975 Ser Arg Arg Lys Tyr Phe Tyr Leu Val Gly
Tyr Gly Met Pro Ala Leu 980 985 990 Ile Val Ala Val Ser Ala Ala Val
Asp Tyr Arg Ser Tyr Gly Thr Asp 995 1000 1005 Lys Val Cys Trp Leu
Arg Leu Asp Thr Tyr Phe Ile Trp Ser Phe 1010 1015 1020 Ile Gly Pro
Ala Thr Leu Ile Ile Met Leu Asn Val Ile Phe Leu 1025 1030 1035 Gly
Ile Ala Leu Tyr Lys Met Phe His His Thr Ala Ile Leu Lys 1040 1045
1050 Pro Glu Ser Gly Cys Leu Asp Asn Ile Lys Ser Trp Val Ile Gly
1055 1060 1065 Ala Ile Ala Leu Leu Cys Leu Leu Gly Leu Thr Trp Ala
Phe Gly 1070 1075 1080 Leu Met Tyr Ile Asn Glu Ser Thr Val
Ile Met Ala Tyr Leu Phe 1085 1090 1095 Thr Ile Phe Asn Ser Leu Gln
Gly Met Phe Ile Phe Ile Phe His 1100 1105 1110 Cys Val Leu Gln Lys
Lys Val Arg Lys Glu Tyr Gly Lys Cys Leu 1115 1120 1125 Arg Thr His
Cys Cys Ser Gly Lys Ser Thr Glu Ser Ser Ile Gly 1130 1135 1140 Ser
Gly Lys Thr Ser Gly Ser Arg Thr Pro Gly Arg Tyr Ser Thr 1145 1150
1155 Gly Ser Gln Ser Arg Ile Arg Arg Met Trp Asn Asp Thr Val Arg
1160 1165 1170 Lys Gln Ser Glu Ser Ser Phe Ile Thr Gly Asp Ile Asn
Ser Ser 1175 1180 1185 Ala Ser Leu Asn Arg Glu Pro Tyr Arg Glu Thr
Ser Met Gly Val 1190 1195 1200 Lys Leu Asn Ile Ala Tyr Gln Ile Gly
Ala Ser Glu Gln Cys Gln 1205 1210 1215 Gly Tyr Lys Cys His Gly Tyr
Ser Thr Thr Glu Trp 1220 1225 1230 9 1527 PRT Rat 9 Met Cys Pro Pro
Gln Leu Phe Ile Leu Met Met Leu Leu Ala Pro Val 1 5 10 15 Val His
Gly Gly Lys His Asn Glu Arg His Pro Ala Leu Ala Ala Pro 20 25 30
Leu Arg His Ala Glu His Ser Pro Gly Gly Pro Leu Pro Pro Arg His 35
40 45 Leu Leu Gln Gln Pro Ala Ala Glu Arg Ser Thr Ala His Arg Gly
Gln 50 55 60 Gly Pro Arg Gly Thr Ala Arg Gly Val Arg Gly Pro Gly
Ala Pro Gly 65 70 75 80 Ala Gln Ile Ala Ala Gln Ala Phe Ser Arg Ala
Pro Ile Pro Met Ala 85 90 95 Val Val Arg Arg Glu Leu Ser Cys Glu
Ser Tyr Pro Ile Glu Leu Arg 100 105 110 Cys Pro Gly Thr Asp Val Ile
Met Ile Glu Ser Ala Asn Tyr Gly Arg 115 120 125 Thr Asp Asp Lys Ile
Cys Asp Ser Asp Pro Ala Gln Met Glu Asn Ile 130 135 140 Arg Cys Tyr
Leu Pro Asp Ala Tyr Lys Ile Met Ser Gln Arg Cys Asn 145 150 155 160
Asn Arg Thr Gln Cys Ala Val Val Ala Gly Pro Asp Val Phe Pro Asp 165
170 175 Pro Cys Pro Gly Thr Tyr Lys Tyr Leu Glu Val Gln Tyr Glu Cys
Val 180 185 190 Pro Tyr Lys Val Glu Gln Lys Val Phe Leu Cys Pro Gly
Leu Leu Lys 195 200 205 Gly Val Tyr Gln Ser Glu His Leu Phe Glu Ser
Asp His Gln Ser Gly 210 215 220 Ala Trp Cys Lys Asp Pro Leu Gln Ala
Ser Asp Lys Ile Tyr Tyr Met 225 230 235 240 Pro Trp Thr Pro Tyr Arg
Thr Asp Thr Leu Thr Glu Tyr Ser Ser Lys 245 250 255 Asp Asp Phe Ile
Ala Gly Arg Pro Thr Thr Thr Tyr Lys Leu Pro His 260 265 270 Arg Val
Asp Gly Thr Gly Phe Val Val Tyr Asp Gly Ala Leu Phe Phe 275 280 285
Asn Lys Glu Arg Thr Arg Asn Ile Val Lys Phe Asp Leu Arg Thr Arg 290
295 300 Ile Lys Ser Gly Glu Ala Ile Ile Ala Asn Ala Asn Tyr His Asp
Thr 305 310 315 320 Ser Pro Tyr Arg Trp Gly Gly Lys Ser Asp Ile Asp
Leu Ala Val Asp 325 330 335 Glu Asn Gly Leu Trp Val Ile Tyr Ala Thr
Glu Gln Asn Asn Gly Lys 340 345 350 Ile Val Ile Ser Gln Leu Asn Pro
Tyr Thr Leu Arg Ile Glu Gly Thr 355 360 365 Trp Asp Thr Ala Tyr Asp
Lys Arg Ser Ala Ser Asn Ala Phe Met Ile 370 375 380 Cys Gly Ile Leu
Tyr Val Val Lys Ser Val Tyr Glu Asp Asp Asp Asn 385 390 395 400 Glu
Ala Thr Gly Asn Lys Ile Asp Tyr Ile Tyr Asn Thr Asp Gln Ser 405 410
415 Lys Asp Ser Leu Val Asp Val Pro Phe Pro Asn Ser Tyr Gln Tyr Ile
420 425 430 Ala Ala Val Asp Tyr Asn Pro Arg Asp Asn Leu Leu Tyr Val
Trp Asn 435 440 445 Asn Tyr His Val Val Lys Tyr Ser Leu Asp Phe Gly
Pro Leu Asp Ser 450 455 460 Arg Ser Gly Pro Val His His Gly Gln Val
Ser Tyr Ile Ser Pro Pro 465 470 475 480 Ile His Leu Asp Ser Asp Leu
Glu Arg Pro Pro Val Arg Gly Ile Ser 485 490 495 Thr Thr Gly Pro Leu
Gly Met Gly Ser Thr Thr Thr Ser Thr Thr Leu 500 505 510 Arg Thr Thr
Thr Trp Asn Leu Gly Arg Ser Thr Thr Pro Ser Leu Pro 515 520 525 Gly
Arg Arg Asn Arg Ser Thr Ser Thr Pro Ser Pro Ala Ile Glu Val 530 535
540 Leu Asp Val Thr Thr His Leu Pro Ser Ala Ala Ser Gln Ile Pro Ala
545 550 555 560 Met Glu Glu Ser Cys Glu Ala Val Glu Ala Arg Glu Ile
Met Trp Phe 565 570 575 Lys Thr Arg Gln Gly Gln Val Ala Lys Gln Ser
Cys Pro Ala Gly Thr 580 585 590 Ile Gly Val Ser Thr Tyr Leu Cys Leu
Ala Pro Asp Gly Ile Trp Asp 595 600 605 Pro Gln Gly Pro Asp Leu Ser
Asn Cys Ser Ser Pro Trp Val Asn His 610 615 620 Ile Thr Gln Lys Leu
Lys Ser Gly Glu Thr Ala Ala Asn Ile Ala Arg 625 630 635 640 Glu Leu
Ala Glu Gln Thr Arg Asn His Leu Asn Ala Gly Asp Ile Thr 645 650 655
Tyr Ser Val Arg Ala Met Asp Gln Leu Val Gly Leu Leu Asp Val Gln 660
665 670 Leu Arg Asn Leu Thr Pro Gly Gly Lys Asp Ser Ala Ala Arg Ser
Leu 675 680 685 Asn Lys Leu Gln Lys Arg Glu Arg Ser Cys Arg Ala Tyr
Val Gln Ala 690 695 700 Met Val Glu Thr Val Asn Asn Leu Leu Gln Pro
Gln Ala Leu Asn Ala 705 710 715 720 Trp Arg Asp Leu Thr Thr Ser Asp
Gln Leu Arg Ala Ala Thr Met Leu 725 730 735 Leu Asp Thr Val Glu Glu
Ser Ala Phe Val Leu Ala Asp Asn Leu Leu 740 745 750 Lys Thr Asp Ile
Val Arg Glu Asn Thr Asp Asn Ile Gln Leu Glu Val 755 760 765 Ala Arg
Leu Ser Thr Glu Gly Asn Leu Glu Asp Leu Lys Phe Pro Glu 770 775 780
Asn Thr Gly His Gly Ser Thr Ile Gln Leu Ser Ala Asn Thr Leu Lys 785
790 795 800 Gln Asn Gly Arg Asn Gly Glu Ile Arg Val Ala Phe Val Leu
Tyr Asn 805 810 815 Asn Leu Gly Pro Tyr Leu Ser Thr Glu Asn Ala Ser
Met Lys Leu Gly 820 825 830 Thr Glu Ala Met Ser Thr Asn His Ser Val
Ile Val Asn Ser Pro Val 835 840 845 Ile Thr Ala Ala Ile Asn Lys Glu
Phe Ser Asn Lys Val Tyr Leu Ala 850 855 860 Asp Pro Val Val Phe Thr
Val Lys His Ile Lys Gln Ser Glu Glu Asn 865 870 875 880 Phe Asn Pro
Asn Cys Ser Phe Trp Ser Tyr Ser Lys Arg Thr Met Thr 885 890 895 Gly
Tyr Trp Ser Thr Gln Gly Cys Arg Leu Leu Thr Thr Asn Lys Thr 900 905
910 His Thr Thr Cys Ser Cys Asn His Leu Thr Asn Phe Ala Val Leu Met
915 920 925 Ala His Val Glu Val Lys His Ser Asp Ala Val His Asp Leu
Leu Leu 930 935 940 Asp Val Ile Thr Trp Val Gly Ile Leu Leu Ser Leu
Val Cys Leu Leu 945 950 955 960 Ile Cys Ile Phe Thr Phe Cys Phe Phe
Arg Gly Leu Gln Ser Asp Arg 965 970 975 Asn Thr Ile His Lys Asn Leu
Cys Ile Ser Leu Phe Val Ala Glu Leu 980 985 990 Leu Phe Leu Ile Gly
Ile Asn Arg Thr Asp Gln Pro Ile Ala Cys Ala 995 1000 1005 Val Phe
Ala Ala Leu Leu His Phe Phe Phe Leu Ala Ala Phe Thr 1010 1015 1020
Trp Met Phe Leu Glu Gly Val Gln Leu Tyr Ile Met Leu Val Glu 1025
1030 1035 Val Phe Glu Ser Glu His Ser Arg Arg Lys Tyr Phe Tyr Leu
Val 1040 1045 1050 Gly Tyr Gly Met Pro Ala Leu Ile Val Ala Val Ser
Ala Ala Val 1055 1060 1065 Asp Tyr Arg Ser Tyr Gly Thr Asp Lys Val
Cys Trp Leu Arg Leu 1070 1075 1080 Asp Thr Tyr Phe Ile Trp Ser Phe
Ile Gly Pro Ala Thr Leu Ile 1085 1090 1095 Ile Met Leu Asn Val Ile
Phe Leu Gly Ile Ala Leu Tyr Lys Met 1100 1105 1110 Phe His His Thr
Ala Ile Leu Lys Pro Glu Ser Gly Cys Leu Asp 1115 1120 1125 Asn Ile
Lys Ser Trp Val Ile Gly Ala Ile Ala Leu Leu Cys Leu 1130 1135 1140
Leu Gly Leu Thr Trp Ala Phe Gly Leu Met Tyr Ile Asn Glu Ser 1145
1150 1155 Thr Val Ile Met Ala Tyr Leu Phe Thr Ile Phe Asn Ser Leu
Gln 1160 1165 1170 Gly Met Phe Ile Phe Ile Phe His Cys Val Leu Gln
Lys Lys Val 1175 1180 1185 Arg Lys Glu Tyr Gly Lys Cys Leu Arg Thr
His Cys Cys Ser Gly 1190 1195 1200 Lys Ser Thr Glu Ser Ser Ile Gly
Ser Gly Lys Thr Ser Gly Ser 1205 1210 1215 Arg Thr Pro Gly Arg Tyr
Ser Thr Gly Ser Gln Ser Arg Ile Arg 1220 1225 1230 Arg Met Trp Asn
Asp Thr Val Arg Lys Gln Ser Glu Ser Ser Phe 1235 1240 1245 Ile Thr
Gly Asp Ile Asn Ser Ser Ala Ser Leu Asn Arg Glu Gly 1250 1255 1260
Leu Leu Asn Asn Ala Arg Asp Thr Ser Val Met Asp Thr Leu Pro 1265
1270 1275 Leu Asn Gly Asn His Gly Asn Ser Tyr Ser Ile Ala Gly Gly
Glu 1280 1285 1290 Tyr Leu Ser Asn Cys Val Gln Ile Ile Asp Arg Gly
Tyr Asn His 1295 1300 1305 Asn Glu Thr Ala Leu Glu Lys Lys Ile Leu
Lys Glu Leu Thr Ser 1310 1315 1320 Asn Tyr Ile Pro Ser Tyr Leu Asn
Asn His Glu Arg Ser Ser Glu 1325 1330 1335 Gln Asn Arg Asn Met Met
Asn Lys Leu Val Asp Asn Leu Gly Ser 1340 1345 1350 Gly Ser Glu Asp
Asp Ala Ile Val Leu Asp Asp Ala Ala Ser Phe 1355 1360 1365 Asn His
Glu Glu Ser Leu Gly Leu Glu Leu Ile His Glu Glu Ser 1370 1375 1380
Asp Ala Pro Leu Leu Pro Pro Arg Val Tyr Ser Thr Asp Asn His 1385
1390 1395 Gln Pro His His Tyr Ser Arg Arg Arg Leu Pro Gln Asp His
Ser 1400 1405 1410 Glu Ser Phe Phe Pro Leu Leu Thr Asp Glu His Thr
Glu Asp Pro 1415 1420 1425 Gln Ser Pro His Arg Asp Ser Leu Tyr Thr
Ser Met Pro Ala Leu 1430 1435 1440 Ala Gly Val Pro Ala Ala Asp Ser
Val Thr Thr Ser Thr Gln Thr 1445 1450 1455 Glu Ala Ala Ala Ala Lys
Gly Gly Asp Ala Glu Asp Val Tyr Tyr 1460 1465 1470 Lys Ser Met Pro
Asn Leu Gly Ser Arg Asn His Val His Pro Leu 1475 1480 1485 His Ala
Tyr Tyr Gln Leu Gly Arg Gly Ser Ser Asp Gly Phe Ile 1490 1495 1500
Val Pro Pro Asn Lys Asp Gly Ala Ser Pro Glu Gly Thr Ser Lys 1505
1510 1515 Gly Pro Ala His Leu Val Thr Ser Leu 1520 1525 10 541 PRT
Homo sapiens 10 Met Asp Phe Glu Ser Gly Gln Val Asp Pro Leu Ala Ser
Val Ile Leu 1 5 10 15 Pro Pro Asn Leu Leu Glu Asn Leu Ser Pro Glu
Asp Ser Val Leu Val 20 25 30 Arg Arg Ala Gln Phe Thr Phe Phe Asn
Lys Thr Gly Leu Phe Gln Asp 35 40 45 Val Gly Pro Gln Arg Lys Thr
Leu Val Ser Tyr Val Met Ala Cys Ser 50 55 60 Ile Gly Asn Ile Thr
Ile Gln Asn Leu Lys Asp Pro Val Gln Ile Lys 65 70 75 80 Ile Lys His
Thr Arg Thr Gln Glu Val His His Pro Ile Cys Ala Phe 85 90 95 Trp
Asp Leu Asn Lys Asn Lys Ser Phe Gly Gly Trp Asn Thr Ser Gly 100 105
110 Cys Val Ala His Arg Asp Ser Asp Ala Ser Glu Thr Val Cys Leu Cys
115 120 125 Asn His Phe Thr His Phe Gly Val Leu Met Asp Leu Pro Arg
Ser Ala 130 135 140 Ser Gln Leu Asp Ala Arg Asn Thr Lys Val Leu Thr
Phe Ile Ser Tyr 145 150 155 160 Ile Gly Cys Gly Ile Ser Ala Ile Phe
Ser Ala Ala Thr Leu Leu Thr 165 170 175 Tyr Val Ala Phe Glu Lys Leu
Arg Arg Asp Tyr Pro Ser Lys Ile Leu 180 185 190 Met Asn Leu Ser Thr
Ala Leu Leu Phe Leu Asn Leu Leu Phe Leu Leu 195 200 205 Asp Gly Trp
Ile Thr Ser Phe Asn Val Asp Gly Leu Cys Ile Ala Val 210 215 220 Ala
Val Leu Leu His Phe Phe Leu Leu Ala Thr Phe Thr Trp Met Gly 225 230
235 240 Leu Glu Ala Ile His Met Tyr Ile Ala Leu Val Lys Val Phe Asn
Thr 245 250 255 Tyr Ile Arg Arg Tyr Ile Leu Lys Phe Cys Ile Ile Gly
Trp Gly Leu 260 265 270 Pro Ala Leu Val Val Ser Val Val Leu Ala Ser
Arg Asn Asn Asn Glu 275 280 285 Val Tyr Gly Lys Glu Ser Tyr Gly Lys
Glu Lys Gly Asp Glu Phe Cys 290 295 300 Trp Ile Gln Asp Pro Val Ile
Phe Tyr Val Thr Cys Ala Gly Tyr Phe 305 310 315 320 Gly Val Met Phe
Phe Leu Asn Ile Ala Met Phe Ile Val Val Met Val 325 330 335 Gln Ile
Cys Gly Arg Asn Gly Lys Arg Ser Asn Arg Thr Leu Arg Glu 340 345 350
Glu Val Leu Arg Asn Leu Arg Ser Val Val Ser Leu Thr Phe Leu Leu 355
360 365 Gly Met Thr Trp Gly Phe Ala Phe Phe Ala Trp Gly Pro Leu Asn
Ile 370 375 380 Pro Phe Met Tyr Leu Phe Ser Ile Phe Asn Ser Leu Gln
Gly Leu Phe 385 390 395 400 Ile Phe Ile Phe His Cys Ala Met Lys Glu
Asn Val Gln Lys Gln Trp 405 410 415 Arg Gln His Leu Cys Cys Gly Arg
Phe Arg Leu Ala Asp Asn Ser Asp 420 425 430 Trp Ser Lys Thr Ala Thr
Asn Ile Ile Lys Lys Ser Ser Asp Asn Leu 435 440 445 Gly Lys Ser Leu
Ser Ser Ser Ser Ile Gly Ser Asn Ser Thr Tyr Leu 450 455 460 Thr Ser
Lys Ser Lys Ser Ser Ser Thr Thr Tyr Phe Lys Arg Asn Ser 465 470 475
480 His Thr Asp Ser Ala Ser Met Asp Lys Ser Leu Ser Lys Leu Ala His
485 490 495 Ala Asp Gly Asp Gln Thr Ser Ile Ile Pro Val His Gln Val
Ile Asp 500 505 510 Lys Val Lys Gly Tyr Cys Asn Ala His Ser Asp Asn
Phe Tyr Lys Asn 515 520 525 Ile Ile Met Ser Asp Thr Phe Ser His Ser
Thr Lys Phe 530 535 540 11 1582 PRT Caenorhabditis elegans 11 Met
Ala Thr Ala Ser Thr Glu Ile Ser Glu Phe Ser Glu Ala Ile Glu 1 5 10
15 Ser Thr Phe Asp Leu Asp Phe Thr Ala His Gln Thr Glu Ile Ile Gly
20 25 30 Thr Tyr Trp Asn Leu Arg Ala Leu Leu Arg Leu His Arg Ser
Leu Val 35 40 45 Ala Ile Asp His Val Ser Gln Lys Ser Phe Trp Glu
Arg Tyr Asn His 50 55 60 Trp Ile Gln Leu Ser Met Leu Val Ser Asn
Gln Asn Val Asn Leu Cys 65 70 75 80 Gln Ser Asn Ile Cys Gln Asn Gly
Gly Thr Cys Leu Val Ala Ser Ser 85 90 95 Val Pro Ala Thr Ala Thr
Cys Pro Lys Asn Ser Ile Tyr Tyr Met Gly 100 105 110 Ser Cys Tyr Val
Phe Asp Thr Thr Leu Arg Asn Trp Asn Asp Ala Ala 115 120 125 Leu Tyr
Cys Asn Asn Met Asn Ser Ala Thr Leu Pro Leu Val Glu Ser 130 135 140
Ala Glu Asp Gln Ala Phe Phe Ala Gly Tyr Leu Gln Ala Met Ile Pro 145
150 155 160 Ser Asn Pro Pro Ala Asp Met Arg Pro Pro Pro Asp Gly Ile
Trp Thr 165 170 175 Ala Val Arg Gly Val Asn Asn Val Thr Arg Ala Ser
Trp Val Tyr Tyr 180 185 190 Pro Gly Ser Phe Leu Val Thr Asp Thr Phe
Trp Ala Pro Gln Glu Pro 195 200 205 Asn Ile Tyr Val Asn Tyr Asn
Asp Val Cys Val Ala Leu Gln Ser Asp 210 215 220 Ser Phe Tyr Arg Glu
Trp Thr Thr Ala Leu Cys Thr Ile Leu Lys Tyr 225 230 235 240 Thr Val
Cys Lys Val Ala Pro Thr Gln Ile Gln Ala Lys Tyr Val Ala 245 250 255
Gln Cys Ser Cys Pro Asn Gly Tyr Gly Gly Gln Thr Cys Glu Thr Gln 260
265 270 Ser Thr Thr Asn Gln Gln Ala Ser Thr Gln Arg Thr Cys Gly Ser
Asn 275 280 285 Asp Phe Gln Phe Ser Cys Pro Asn Asp Gln Thr Ile Thr
Val Asp Phe 290 295 300 Ala Ser Phe Gly Ala Gln Gly Gly Ser Ile Ile
Thr Ser Pro Pro Asp 305 310 315 320 Ala Leu Leu Gln Gln Ile Val Gln
Lys Val Asn Ala Glu Thr Lys Lys 325 330 335 Thr Val Asn Phe Trp Ile
Gly Thr Pro Asn Asn Cys Gln Leu Leu Met 340 345 350 Val Thr Gly Ser
Ser Thr Ser Tyr Ser Gln Cys Pro Ser Ser Pro Ser 355 360 365 Ser Thr
Ala Asn Val Ile Cys Ser Thr Val Pro Gln Ser Thr Ala Ser 370 375 380
Val Ser Ala Arg Pro Thr Gln Ser Ala Pro Val Asp Pro Val Ser Gln 385
390 395 400 Thr Met Ala Arg Arg Glu Val Tyr Thr Gly Val Gln Pro Ile
Ala Ser 405 410 415 Ala Leu Gly Gly Gln Ser Lys Lys Thr Asn Arg Lys
Leu Asn Asn Ile 420 425 430 Cys Gln Thr Lys Ile Gly Ala Pro Leu Ser
Leu Phe Leu Phe Ser Arg 435 440 445 Asn Glu Val Ile Thr Gly Phe Val
Cys Ile Ser Leu Ile Ser Ala Ser 450 455 460 Pro Gln Ile Ile Tyr Tyr
Leu Cys Ala Val Ser Leu Ile Cys His Pro 465 470 475 480 Ser Val Pro
Asp Ser Ile Asn Lys Pro Arg Tyr Cys Lys Lys Glu Lys 485 490 495 Lys
Asp Gly Ile Thr Tyr Glu Gln Thr Arg Ala Cys Met Leu His Glu 500 505
510 Gln Pro Cys Pro Asp Pro Gln Asn Val Glu Gly Thr Val Thr Arg Tyr
515 520 525 Cys Asn Cys Gln Thr Ala Lys Trp Glu Thr Pro Asp Thr Thr
Asn Cys 530 535 540 Thr His Arg Trp Val Ala Glu Met Glu Thr Ala Ile
Lys Asp Asn Gln 545 550 555 560 Pro Val Glu Asp Ile Ser Ser Thr Val
Asn Arg Gln Leu Lys Ser Thr 565 570 575 Ile Glu Arg Thr Leu Phe Gly
Gly Asp Ile Thr Gly Thr Val Arg Leu 580 585 590 Ser Asn Asp Met Leu
Ser Leu Ala Arg Asn Gln Phe Ser Val Leu Asn 595 600 605 Asp Arg Asn
Leu Arg Glu Asn Lys Ala Arg Asn Phe Thr Glu Asn Leu 610 615 620 Gly
Gly Ser Gly Asp Gln Leu Leu Ser Pro Val Ala Ala Thr Val Trp 625 630
635 640 Asp Gln Leu Ser Ser Thr Ile Arg Ile Gln His Ala Ser Lys Leu
Met 645 650 655 Ser Val Leu Glu Gln Ser Val Leu Leu Leu Gly Asp Tyr
Met Thr Asp 660 665 670 Gln Lys Leu Asn Leu Gln Tyr Ile Asn Trp Ala
Met Glu Val Glu Arg 675 680 685 Ser Glu Pro Glu Val Gln Thr Phe Gly
Ala Ala Ala Ser Pro Asn Val 690 695 700 Gln Asp Asp Met Gly Met Met
Arg Val Met Ala Ala Ala Pro Pro Ala 705 710 715 720 Pro Gln Pro Glu
Thr Asn Thr Thr Ile Met Phe Pro Ser Leu Lys Leu 725 730 735 Ser Pro
Thr Ile Thr Leu Pro Ser Ala Ser Leu Leu Ser Ser Leu Ala 740 745 750
Ser Pro Thr Pro Val Ala Gly Gly Gly Pro Ser Ile Leu Ser Ser Phe 755
760 765 Gln Asp Asp Thr Pro Val Gly Met Ala Ser Thr Pro Asn Leu Asn
Arg 770 775 780 Asn Pro Val Lys Leu Gly Tyr Tyr Ala Phe Ala Gly Phe
Gly Gln Leu 785 790 795 800 Leu Asn Asn Asn Asn Asp His Thr Leu Ile
Asn Ser Gln Val Ile Gly 805 810 815 Ala Ser Ile Gln Asn Ala Thr Gln
Ser Val Thr Leu Pro Val Asp His 820 825 830 Pro Val Thr Phe Thr Phe
Gln His Leu Thr Thr Lys Gly Val Ser Asn 835 840 845 Pro Arg Cys Val
Tyr Trp Asp Leu Met Glu Ser Lys Trp Ser Thr Leu 850 855 860 Gly Cys
Thr Leu Ile Ala Thr Ser Ser Asn Ser Ser Gln Cys Ser Cys 865 870 875
880 Thr His Leu Thr Ser Phe Ala Ile Leu Met Asp Ile Ser Gly Gln Val
885 890 895 Gly Arg Leu Ser Gly Gly Leu Ala Ser Ala Leu Asp Val Val
Ser Thr 900 905 910 Ile Gly Cys Ala Ile Ser Ile Val Cys Leu Ala Leu
Ser Val Cys Val 915 920 925 Phe Thr Phe Phe Arg Asn Leu Gln Asn Val
Arg Asn Ser Ile His Arg 930 935 940 Asn Leu Cys Leu Cys Leu Leu Ile
Ala Glu Leu Val Phe Val Ile Gly 945 950 955 960 Met Asp Arg Thr Gly
Asn Arg Thr Gly Cys Gly Val Val Ala Ile Leu 965 970 975 Leu His Tyr
Phe Phe Leu Ser Ser Phe Cys Trp Met Leu Leu Glu Gly 980 985 990 Tyr
Gln Leu Tyr Met Met Leu Ile Gln Val Phe Glu Pro Asn Arg Thr 995
1000 1005 Arg Ile Phe Leu Tyr Tyr Leu Phe Cys Tyr Gly Thr Pro Ala
Val 1010 1015 1020 Val Val Ala Ile Ser Ala Gly Ile Lys Trp Glu Asp
Tyr Gly Thr 1025 1030 1035 Asp Ser Tyr Cys Trp Ile Asp Thr Ser Thr
Pro Thr Ile Trp Ala 1040 1045 1050 Phe Val Ala Pro Ile Ile Val Ile
Ile Ala Ala Asn Ile Ile Phe 1055 1060 1065 Leu Leu Ile Ala Leu Lys
Val Val Leu Ser Val Gln Ser Arg Asp 1070 1075 1080 Arg Thr Lys Trp
Gly Arg Ile Ile Gly Trp Leu Lys Gly Ser Ala 1085 1090 1095 Thr Leu
Leu Cys Leu Leu Gly Ile Thr Trp Ile Phe Gly Phe Leu 1100 1105 1110
Thr Ala Val Lys Gly Gly Thr Gly Thr Ala Phe Ala Trp Ile Phe 1115
1120 1125 Thr Ile Leu Asn Cys Thr Gln Gly Ile Phe Ile Phe Val Leu
His 1130 1135 1140 Val Val Leu Asn Glu Lys Val Arg Ala Ser Ile Val
Arg Trp Leu 1145 1150 1155 Arg Thr Gly Ile Cys Cys Leu Pro Glu Thr
Ser Ser Ala Ala Tyr 1160 1165 1170 Asn Ser Arg Ser Phe Leu Ser Ser
Arg Gln Arg Ile Leu Asn Met 1175 1180 1185 Ile Lys Val Asn Gly His
Ser Tyr Pro Ser Thr Ala Ser Thr Asp 1190 1195 1200 Asp Lys Glu Lys
Gln Leu Thr Pro Ile Thr Lys Thr Thr Asp Trp 1205 1210 1215 Leu Ser
Arg Leu Pro Asn Gln Asp Ser Val Ser Ile Pro Glu Ser 1220 1225 1230
Asn Phe Asn Asn Leu Asn Gly Thr Leu Glu Asn Ser Asn Leu Asn 1235
1240 1245 Ser Ala Glu Ile Lys Glu Glu Asp Glu Ile Pro Glu Leu Arg
Arg 1250 1255 1260 Arg Val Thr Val Asp Leu Asn Pro Met Ile Val Ser
Asn Asn Glu 1265 1270 1275 Ile Glu Arg Met Ser His Ala Ser Ser Asp
Pro Arg Gly Ser Gln 1280 1285 1290 Ile Ile Glu Val Thr Ala Val Glu
Lys Lys Ala Pro Val Lys Arg 1295 1300 1305 Ile Lys Phe Pro Leu Gly
Ala Lys Gln Ser Glu Arg Gly Ser Gln 1310 1315 1320 His Arg Thr Lys
Ala Lys His Gly Thr Gly Thr Leu Val Ser Pro 1325 1330 1335 Trp His
Ile Val Thr Ala Ala His Leu Ile Gly Ile Ser Glu Asp 1340 1345 1350
Pro Leu Pro Asp Cys Asp Thr Gly Asn Leu Arg Glu Ala Tyr Phe 1355
1360 1365 Val Arg Asp Tyr Lys Asn Phe Val Ala Phe Val Asn Val Thr
Cys 1370 1375 1380 Ala Val Pro Glu Met Cys Lys Gly Leu His Arg Lys
Asp Met Phe 1385 1390 1395 Lys Pro Leu Ala Ile Lys Ser Leu Tyr Ile
Arg Lys Gly Tyr Val 1400 1405 1410 Gly Asp Gly Cys Ile Asp Arg Glu
Ser Phe Asn Asp Ile Ala Val 1415 1420 1425 Phe Glu Leu Glu Glu Pro
Ile Glu Phe Ser Lys Asp Ile Phe Pro 1430 1435 1440 Ala Cys Leu Pro
Ser Ala Pro Lys Ile Pro Arg Ile Arg Glu Thr 1445 1450 1455 Gly Tyr
Lys Leu Phe Gly Tyr Gly Arg Asp Pro Ser Asp Ser Val 1460 1465 1470
Leu Glu Ser Gly Lys Leu Lys Ser Leu Tyr Ser Phe Val Ala Glu 1475
1480 1485 Cys Ser Asp Asp Phe Pro Tyr Gly Gly Val Tyr Cys Thr Ser
Ala 1490 1495 1500 Val Asn Arg Gly Leu Ser Cys Asp Gly Asp Ser Gly
Ser Gly Val 1505 1510 1515 Val Arg Thr Ser Asp Thr Arg Asn Val Gln
Val Leu Val Gly Val 1520 1525 1530 Leu Ser Ala Gly Met Pro Cys Pro
Glu Leu Tyr Asp Thr His Asn 1535 1540 1545 Arg Gln Arg Gln Gln Arg
Arg Gln Leu Thr Gln Glu Thr Asp Leu 1550 1555 1560 Leu Val Asp Val
Ser Ala His Val Asp Phe Phe Cys Thr Cys Cys 1565 1570 1575 Gly Met
Cys Ser 1580 12 198 PRT Homo sapiens 12 Met Glu Thr Tyr Ser Leu Ser
Leu Gly Asn Gln Ser Val Val Glu Pro 1 5 10 15 Asn Ile Ala Ile Gln
Ser Ala Asn Phe Ser Ser Glu Asn Ala Val Gly 20 25 30 Pro Ser Asn
Val Arg Phe Ser Val Gln Lys Gly Ala Ser Ser Ser Leu 35 40 45 Val
Ser Ser Ser Thr Phe Ile His Thr Asn Val Asp Gly Leu Asn Pro 50 55
60 Asp Ala Gln Thr Glu Leu Gln Val Leu Leu Asn Met Thr Lys Asn Tyr
65 70 75 80 Thr Lys Thr Cys Gly Phe Val Val Tyr Gln Asn Asp Lys Leu
Phe Gln 85 90 95 Ser Lys Thr Phe Thr Ala Lys Ser Asp Phe Ser Gln
Lys Ile Ile Ser 100 105 110 Ser Lys Thr Asp Glu Asn Glu Gln Asp Gln
Ser Ala Ser Val Asp Met 115 120 125 Val Phe Ser Pro Lys Tyr Asn Gln
Lys Glu Phe Gln Leu Tyr Ser Tyr 130 135 140 Ala Cys Val Tyr Trp Asn
Leu Ser Ala Lys Asp Trp Asp Thr Tyr Gly 145 150 155 160 Cys Gln Lys
Asp Lys Gly Thr Asp Gly Phe Leu Arg Cys Arg Cys Asn 165 170 175 His
Thr Thr Asn Phe Ala Val Leu Met Thr Phe Lys Lys Asp Tyr Gln 180 185
190 Tyr Pro Lys Ser Leu Asp 195 13 10 PRT Homo sapiens 13 Gln Ile
Val Thr Arg Lys Val Arg Lys Thr 1 5 10 14 38 PRT Homo sapiens 14
Glu Asn Ser Asn Lys Asn Leu Gln Thr Ser Asp Gly Asp Ile Asn Asn 1 5
10 15 Ile Asp Phe Asp Asn Asn Asp Ile Pro Arg Thr Asp Thr Ile Asn
Ile 20 25 30 Pro Asn Pro Met Cys Thr 35 15 10 PRT Homo sapiens 15
Ile Arg Thr Met Lys Pro Leu Pro Arg His 1 5 10 16 41 PRT Homo
sapiens 16 Thr Val Gly Val Ile Tyr Ser Gln Asn Gly Asn Asn Pro Gln
Trp Glu 1 5 10 15 Leu Asp Tyr Arg Gln Glu Lys Ile Cys Trp Leu Ala
Ile Pro Glu Pro 20 25 30 Asn Gly Val Ile Lys Ser Pro Leu Leu 35 40
17 25 PRT Homo sapiens 17 Thr Ile Ser Ile Lys Val Leu Trp Lys Asn
Asn Gln Asn Leu Thr Ser 1 5 10 15 Thr Lys Lys Val Ser Ser Met Lys
Lys 20 25 18 6 PRT Homo sapiens 18 Asn Asp Asp Ser Ile Arg 1 5 19
78 PRT Homo sapiens 19 Tyr Thr Val Arg Thr Lys Val Phe Gln Ser Glu
Ala Ser Lys Val Leu 1 5 10 15 Met Leu Leu Ser Ser Ile Gly Arg Arg
Lys Ser Leu Pro Ser Val Thr 20 25 30 Arg Pro Arg Leu Arg Val Lys
Met Tyr Asn Phe Leu Arg Ser Leu Pro 35 40 45 Thr Leu His Glu Arg
Phe Arg Leu Leu Glu Thr Ser Pro Ser Thr Glu 50 55 60 Glu Ile Thr
Leu Ser Glu Ser Asp Asn Ala Lys Glu Ser Ile 65 70 75 20 38 DNA
Artificial Sequence HGPRBMY6 5' PRIMER 20 cgggatgcct agatgctttc
ctttgcattg tcactttc 38 21 66 DNA Artificial Sequence HGPRBMY6 3'
FLAG TAG PRIMER 21 cggggatccc tacttgtcgt cgtcgtcctt gtagtccatg
atgctttcct ttgcattgtc 60 actttc 66 22 23 DNA Artificial Sequence
HGPRBMY6 Forward primer 383 22 cagacaccat taacatcccg aat 23 23 22
DNA Artificial Sequence HGPRBMY6 Reverse primer 384 23 agaatgaaat
gccgaggaag ag 22 24 17 DNA Artificial Sequence GAPDH-F3 forward
primer 24 agccgagcca catcgct 17 25 19 DNA Artificial Sequence
GAPDH-R1 reverse primer 25 gtgaccaggc gcccaatac 19 26 28 DNA Homo
sapiens 26 caaatccgtt gactccgacc ttcacctt 28 27 13 PRT Homo sapiens
27 Gln Ser Lys Thr Phe Thr Ala Lys Ser Asp Phe Ser Gln 1 5 10 28 13
PRT Homo sapiens 28 Ala Lys Ser Asp Phe Ser Gln Lys Ile Ile Ser Ser
Lys 1 5 10 29 13 PRT Homo sapiens 29 Ser Gln Lys Ile Ile Ser Ser
Lys Thr Asp Glu Asn Glu 1 5 10 30 13 PRT Homo sapiens 30 Val Asp
Met Val Phe Ser Pro Lys Tyr Asn Gln Lys Glu 1 5 10 31 13 PRT Homo
sapiens 31 Val Tyr Trp Asn Leu Ser Ala Lys Asp Trp Asp Thr Tyr 1 5
10 32 13 PRT Homo sapiens 32 Phe Ala Val Leu Met Thr Phe Lys Lys
Asp Tyr Gln Tyr 1 5 10 33 13 PRT Homo sapiens 33 Ile Phe Gln Ile
Val Thr Arg Lys Val Arg Lys Thr Ser 1 5 10 34 13 PRT Homo sapiens
34 Phe Gly Ile Glu Asn Ser Asn Lys Asn Leu Gln Thr Ser 1 5 10 35 13
PRT Homo sapiens 35 Tyr Leu Leu Ile Arg Thr Met Lys Pro Leu Pro Arg
His 1 5 10 36 13 PRT Homo sapiens 36 Met Phe Ile Thr Ile Ser Ile
Lys Val Leu Trp Lys Asn 1 5 10 37 13 PRT Homo sapiens 37 Asn Gln
Asn Leu Thr Ser Thr Lys Lys Val Ser Ser Met 1 5 10 38 13 PRT Homo
sapiens 38 Gln Asn Leu Thr Ser Thr Lys Lys Val Ser Ser Met Lys 1 5
10 39 13 PRT Homo sapiens 39 Thr Lys Lys Val Ser Ser Met Lys Lys
Ile Val Ser Thr 1 5 10 40 13 PRT Homo sapiens 40 Leu Val Asn Asp
Asp Ser Ile Arg Ile Val Phe Ser Tyr 1 5 10 41 13 PRT Homo sapiens
41 Ile Phe Ile Leu Tyr Thr Val Arg Thr Lys Val Phe Gln 1 5 10 42 14
PRT Homo sapiens 42 Ser Leu Gly Asn Gln Ser Val Val Glu Pro Asn Ile
Ala Ile 1 5 10 43 14 PRT Homo sapiens 43 Ser Thr Phe Ile His Thr
Asn Val Asp Gly Leu Asn Pro Asp 1 5 10 44 14 PRT Homo sapiens 44
Gln Lys Ile Ile Ser Ser Lys Thr Asp Glu Asn Glu Gln Asp 1 5 10 45
14 PRT Homo sapiens 45 Val Tyr Trp Asn Leu Ser Ala Lys Asp Trp Asp
Thr Tyr Gly 1 5 10 46 14 PRT Homo sapiens 46 Lys Asn Leu Gln Thr
Ser Asp Gly Asp Ile Asn Asn Ile Asp 1 5 10 47 14 PRT Homo sapiens
47 Leu Arg Ser Leu Pro Thr Leu His Glu Arg Phe Arg Leu Leu 1 5 10
48 14 PRT Homo sapiens 48 Leu Glu Thr Ser Pro Ser Thr Glu Glu Ile
Thr Leu Ser Glu 1 5 10 49 14 PRT Homo sapiens 49 Ser Thr Glu Glu
Ile Thr Leu Ser Glu Ser Asp Asn Ala Lys 1 5 10 50 14 PRT Homo
sapiens 50 Glu Glu Ile Thr Leu Ser Glu Ser Asp Asn Ala Lys Glu Ser
1 5 10 51 14 PRT Homo sapiens 51 Val Thr Arg Lys Val Arg Lys Thr
Ser Val Thr Trp Val Leu 1 5 10 52 14 PRT Homo sapiens 52 Asn Leu
Thr Ser Thr Lys Lys Val Ser Ser Met Lys Lys Ile 1 5 10 53 14 PRT
Homo sapiens 53 Leu Ser Ser Ile Gly Arg Arg Lys Ser Leu Pro Ser Val
Thr 1 5 10 54 14 PRT Homo sapiens 54 Ser Leu Ser Leu Gly Asn Gln
Ser Val Val Glu Pro Asn Ile 1 5 10 55 14 PRT Homo sapiens 55 Ala
Ile Gln Ser Ala Asn Phe Ser Ser Glu Asn Ala Val Gly 1 5 10 56 14
PRT Homo sapiens 56 Leu Gln Val Leu Leu Asn Met Thr Lys Asn Tyr Thr
Lys Thr 1 5 10 57 14 PRT Homo sapiens 57 Leu Asn Met Thr Lys Asn
Tyr Thr Lys Thr Cys Gly Phe Val 1 5 10 58 14 PRT Homo sapiens 58
Ala Cys Val Tyr Trp Asn
Leu Ser Ala Lys Asp Trp Asp Thr 1 5 10 59 14 PRT Homo sapiens 59
Leu Arg Cys Arg Cys Asn His Thr Thr Asn Phe Ala Val Leu 1 5 10 60
14 PRT Homo sapiens 60 Trp Lys Asn Asn Gln Asn Leu Thr Ser Thr Lys
Lys Val Ser 1 5 10 61 14 PRT Homo sapiens 61 Ile Phe Cys Leu Phe
Asn Thr Thr Gln Gly Leu Gln Ile Phe 1 5 10 62 16 PRT Homo sapiens
62 Phe Ser Val Gln Lys Gly Ala Ser Ser Ser Leu Val Ser Ser Ser Thr
1 5 10 15 63 16 PRT Homo sapiens 63 Ile Leu Ser Asn Val Gly Cys Ala
Leu Ser Val Thr Gly Leu Ala Leu 1 5 10 15 64 16 PRT Homo sapiens 64
Ala Leu Ser Val Thr Gly Leu Ala Leu Thr Val Ile Phe Gln Ile Val 1 5
10 15 65 16 PRT Homo sapiens 65 Leu Leu Phe Val Phe Gly Ile Glu Asn
Ser Asn Lys Asn Leu Gln Thr 1 5 10 15 66 16 PRT Homo sapiens 66 Val
Ala Ile Thr Val Gly Val Ile Tyr Ser Gln Asn Gly Asn Asn Pro 1 5 10
15 67 99 DNA Artificial Sequence Randomized Synthetic Oligo 67
cgaagcgtaa gggcccagcc ggccnnknnk nnknnknnkn nknnknnknn knnknnknnk
60 nnknnknnkn nknnknnknn knnkccgggt ccgggcggc 99 68 98 DNA
Artificial Sequence Randomized Synthetic Oligo 68 aaaaggaaaa
aagcggccgc vnnvnnvnnv nnvnnvnnvn nvnnvnnvnn vnnvnnvnnv 60
nnvnnvnnvn nvnnvnnvnn vnngccgccc ggacccgg 98 69 5 PRT Artificial
Sequence Synthetic Polypeptide 69 Pro Gly Pro Gly Gly 1 5 70 15 PRT
Artificial Sequence Synthetic Polypeptide 70 Phe Ala Gly Gln Ile
Ile Trp Tyr Asp Ala Leu Asp Thr Leu Met 1 5 10 15 71 15 PRT
Artificial Sequence Synthetic Polypeptide 71 Ser Asp Phe Val Gly
Gly Phe Trp Phe Trp Asp Ser Leu Phe Asn 1 5 10 15 72 15 PRT
Artificial Sequence Synthetic Polypeptide 72 Gly Asp Phe Trp Tyr
Glu Ala Cys Glu Ser Ser Cys Ala Phe Trp 1 5 10 15 73 15 PRT
Artificial Sequence Synthetic Polypeptide 73 Leu Glu Trp Gly Ser
Asp Val Phe Tyr Asp Val Tyr Asp Cys Cys 1 5 10 15 74 14 PRT
Artificial Sequence Synthetic Polypeptide 74 Arg Ile Asp Ser Cys
Ala Lys Tyr Phe Leu Arg Ser Cys Asp 1 5 10 75 15 PRT Artificial
Sequence Synthetic Polypeptide 75 Cys Leu Arg Ser Gly Thr Gly Cys
Ala Phe Gln Leu Tyr Arg Phe 1 5 10 15 76 15 PRT Artificial Sequence
Synthetic Polypeptide 76 Phe Arg Val Ser Arg Val Trp Asn Pro Pro
Ser Phe Asp Ser Ala 1 5 10 15 77 15 PRT Artificial Sequence
Synthetic Polypeptide 77 His Ala Tyr Val Glu Cys Asn Asp Thr Asp
Cys Arg Val Trp Phe 1 5 10 15 78 39 DNA Artificial Sequence
Synthetic 5' Primer 78 gcagcagcgg ccgcgacata ttatccaacg ttggatgtg
39 79 35 DNA Artificial Sequence Synthetic 3' Primer 79 gcagcagtcg
acgatgcttt cctttgcatt gtcac 35 80 39 DNA Artificial Sequence
Synthetic 5' Primer 80 gcagcagcgg ccgcatggag acttattcct tgtctttgg
39 81 37 DNA Artificial Sequence Synthetic 3' Primer 81 gcagcagtcg
acgtacagga taaaaatttg caatccc 37
* * * * *
References